Functional competence of a partially engaged

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Received 18 Apr 2016 | Accepted 30 Sep 2016 | Published 9 Nov 2016

Punita Kumari1, Ashish Srivastava1, Ramanuj Banerjee1, Eshan Ghosh1, Pragya Gupta1, Ravi Ranjan1, Xin Chen2, Bhagyashri Gupta1, Charu Gupta1, Deepika Jaiman1 & Arun K. Shukla1

GPCRs docks to the N-domain of barr rst and then seven transmembrane core of the receptor engages with barr. It is currently unknown whether fully engaged GPCRbarr complex is essential for functional outcomes or partially engaged complex can also be functionally competent. Here we assemble partially and fully engaged complexes of a chimeric b2V2R with barr1, and discover that the core interaction is dispensable for receptor endocytosis, ERK MAP kinase binding and activation. Furthermore, we observe that carvedilol, a barr biased ligand, does not promote detectable engagement between barr1 and the receptor core. These ndings uncover a previously unknown aspect of GPCR-barr interaction and provide novel insights into GPCR signalling and regulatory paradigms.

DOI: 10.1038/ncomms13416 OPEN

Functional competence of a partially engaged

GPCRb-arrestin complex

1 Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India. 2 School of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou, Jiangsu 213164, China. Correspondence and requests for materials should be addressed to A.K.S. (email: mailto:[email protected]

Web End [email protected] ).

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Gprotein-coupled receptor (GPCR) family consists of B800 different members that exhibit a highly conserved seven transmembrane architecture1. GPCRs bind to

an incredibly diverse range of ligands, still, their signalling and regulatory mechanisms are primarily conserved2. GPCR signalling and downregulation is critically mediated by barrs which on one hand, terminate G protein coupling presumably by steric hindrance and on the other, initiate G protein independent signalling cascades3. There has been a remarkable progress in structural visualization of GPCRs in the recent years4. However, structural details of GPCRbarr interaction have just started to emerge and still remain in infancy. Interaction of the N-domain of arrestins with phosphorylated carboxyl terminus of GPCRs is the rst step in receptor-arrestin binding. Interestingly, a number of biophysical studies using spectroscopy approaches have suggested the engagement of different arrestin loops with the activated receptor core as the second step of interaction58. Crystal structure of rhodopsin with isolated nger loop peptide has directly established a binding interface between the receptor core and the nger loop of visual arrestin9. Recently determined crystal structure of rhodopsin-arrestin complex also exhibits an engagement of the receptor core with arrestin10 although the carboxyl terminus of rhodopsin in this complex is covalently fused and not phosphorylated. Recent visualization of b2V2Rbarr1 complex by negative stain electron microscopy and cross-linking has directly demonstrated a biphasic mechanism of GPCRbarr interaction11. In the rst step, the phosphorylated carboxyl terminus of GPCRs interacts with the N-domain of barrs and in the second step, barrs engage with the cytoplasmic surface of the transmembrane bundle of the receptor (that is, receptor core) (Fig. 1a).

The functional repertoire of GPCR-barr signalling axis is quite broad and spans a wide range of cellular and physiological processes3,1214. This is primarily mediated by a large number of interactions of barrs and their abilities to scaffold a wide array of kinases and other signalling molecules12,13. However, the structural and mechanistic requirements for such a broad functional coverage of GPCRbarr interaction remains currently unexplored. In particular, whether a fully engaged GPCRbarr complex is essential for triggering downstream functional outcomes or even partially engaged complexes might display functional competence remains currently unknown. Phosphorylation of the carboxyl terminus of GPCRs is the primary determinant for barr interaction and this rst step of biphasic interaction represents the high-afnity component in GPCRbarr complex1517. Direct visualization of a partially engaged b2V2Rbarr1 complex11 associated solely through the phosphorylated carboxyl terminus of the receptor by electron microscopy suggests that core interaction may be dispensable for stable assembly of the complex. However, functional capabilities of such a partially engaged receptorbarr complex remain currently unexplored.

Accordingly, here we set out to investigate whether a b2V2Rbarr1 complex associated only through the phosphorylated carboxyl terminus of the receptor and lacking the core interaction might be functionally competent. We focus on recruitment and activation of ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinase, a readout that has become quintessential for barr mediated GPCR signalling, and receptor endocytosis. We assemble partially and fully engaged b2V2Rbarr1 complexes, validate them by uorescence spectroscopy and discover, in contrast with generally believed notion, that the core interaction in this complex is dispensable for ERK2 binding and activation. We also nd that a receptor mutant lacking the core interaction with barr efciently undergoes agonist promoted internalization. Moreover, we also

discover that a barr biased ligand does not promote core interaction between the receptor and barr.

ResultsPartially and fully engaged b2V2Rbarr1 complexes.

Reconstitution of a stable and functional GPCRbarr complex for biophysical studies still remains very challenging. Recently, a strategy has been described for the isolation of a stable barr1 complex with a chimeric b2 adrenergic receptor (b2AR)

harbouring the carboxyl terminus of the arginine vasopressin subtype 2 receptor (V2R), referred to as b2V2R (ref. 11). b2V2R displays b2AR pharmacology but tighter binding with barr18. Stable b2V2Rbarr1 complex can be isolated through coexpression of the receptor and barr1 in cells followed by stabilization using a synthetic antibody fragment (referred to as Fab30) (ref. 11). In order to make this strategy more versatile and amenable to direct biophysical studies, we rst assessed the feasibility of b2V2Rbarr1-Fab30 complex assembly using puried components in-vitro (Fig. 1b). We immobilized puried Fab30 (Supplementary Fig. 1 and Supplementary Fig. 2A) on a polystyrene surface (MaxiSorp 96 well plate) as an anchor to stabilize the complex followed by addition of puried barr1 and

N-terminally FLAG-tagged b2V2R (Supplementary Fig. 2B and Supplementary Fig. 3). After rigorous washing of the surface, we visualized the assembly of the complex using HRP-coupled anti-FLAG M2 antibody. We observed a robust assembly of b2V2Rbarr1 complex that is sensitive to agonist occupancy and phosphorylation status of the receptor, suggesting the formation of a cellularly and pharmacologically relevant complex (Fig. 1c and Supplementary Fig. 2CE).

As mentioned earlier, in biphasic GPCRbarr interaction, the rst step depends primarily on phosphorylation of carboxyl terminus of the receptor while the second step requires an activated receptor core (that is, transmembrane bundle). Therefore, in order to generate partially and fully engaged complexes, we designed an experimental scheme (Fig. 1d) where we trigger receptor phosphorylation in cells by stimulating them with a low-afnity full agonist, isoproterenol and then wash off the agonist in subsequent purication steps. This leads to purication of ligand free b2V2R with phosphorylated carboxyl terminus (referred to as Apob2V2Rphos). Subsequent incubation with high-afnity partial inverse agonist (carazolol) or high-afnity full agonist (BI-167107) results in Inactb2V2Rphos (inactive receptor core with phosphorylated carboxyl terminus) and

actb2V2Rphos (active receptor core with phosphorylated carboxyl terminus), respectively. These two species of the b2V2R provide us a handle to assemble partially (that is, tail only engaged) and fully (that is, tail core engaged) associated b2V2Rbarr1

complexes and evaluate their functional competence in-vitro. As presented in Fig. 1e,f, both, the Inactb2V2Rphos and the

actb2V2Rphos exhibited robust complex formation with barr1 and presumably represent, partially and fully engaged b2V2Rbarr1 complexes, respectively.

In order to conrm the nature of these complexes with respect to tail and core engagement, we utilized a bimane uorescence spectroscopy approach. Extensive previous studies have used bimane labelling in the nger loop of visual arrestin to study its interaction with rhodopsin and reported that rhodopsin-arrestin interaction leads to a signicant decrease in bimane uorescence5,9,19,20. Direct engagement of nger loop of visual arrestin with rhodopsin has also been documented by NMR21 and crystallography9. Crystal structure of rhodopsin-arrestin complex also reveals an engagement of the nger loop of visual arrestin with the transmembrane core of rhodopsin10. More recently, chemical cross-linking and structural modelling of

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13416 ARTICLE

HRP

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Figure 1 | Assembly of partially and fully engaged b2V2Rbarr1-Fab30 complex. (a) Schematic representation of biphasic GPCRbarr interaction. barr interacts with activated and phosphorylated GPCRs in a biphasic fashion where the rst step is binding of barr through the phosphorylated carboxyl terminus and the second step is the engagement of barr with the 7TM core of the receptor. The receptor component is shown in grey, phosphorylated carboxyl terminus in yellow and barr 1 in blue/magenta. (b) Schematic representation of an ELISA-based approach for in-vitro assembly of b2V2Rbarr1

complex. Puried Fab30 is immobilized on solid support as an anchor to capture the complex followed by incubation with puried b2V2R and barr1. Formation of b2V2Rbarr1 complex is visualized using HRP-coupled anti-FLAG M2 antibody through detection of FLAG tagged b2V2R. (c) Fab 30 assisted in-vitro assembly of b2V2Rbarr1 complex. Agonist bound and phosphorylated b2V2R (Actb2V2Rphos) forms a stable complex while inverse agonist bound and non-phosphorylated b2V2R (Inactb2V2Rnon-phos) does not exhibit any detectable complex formation. (d) An experimental set-up to assemble tail only

engaged and fully engaged b2V2Rbarr1 complex in-vitro. b2V2R is coexpressed with GRK2CAAX in cultured Sf9 cells and 66 h post-infection, cells are stimulated with a low-afnity agonist (Isoproterenol) to trigger receptor phosphorylation. Subsequently, the receptor is puried by afnity chromatography and the ligand is washed off during purication to yield ligand free phosphorylated b2V2R (Apob2V2Rphos). Incubation with inverse agonist (carazolol) or high-afnity full agonist (BI-167107) yields Inactb2V2Rphos and Actb2V2Rphos, respectively. (e) Both, the Inactb2V2Rphos and Actb2V2Rphos form a stable complex with barr1 as assessed by ELISA approach and potentially represent tail only and fully engaged complexes, respectively. (f) Formation of tail only engaged and fully engaged complexes as assessed by coimmunoprecipitation experiment. This experiment was repeated three times with identical results and a representative image is shown. Signals in c and e are normalized with Actb2V2Rphos barr1 Fab30 condition as 100%. Data presented in c

and e represent means.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (***Po0.001).

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b2V2Rbarr1 complex has also identied the nger loop of barr1 (residues 62-72) as a major interaction interface with the seven transmembrane core of the receptor11 (Fig. 2a). Therefore, we rst designed a cysteine-less barr1 mutant and then exchanged Leu68 in the nger loop with a cysteine (referred to as barrL68C). We selected L68C based on previous studies with rhodopsin-visual arrestin system that have used the corresponding position (L72C) in the nger loop5,6,19,20,22. We subsequently puried barr1L68C and labelled it with an environmentally sensitive uorophore monobromobimane (mBBr) at Cys68. Based on rhodopsin-arrestin studies, we reasoned that the environment of mBBr should change upon the engagement of the nger loop with the receptor core and, therefore, a change in mBBr uorescence intensity will reect the core interaction between b2V2R and barr1. We conrmed the functionality of mBBr-labelled barr1 with respect to its binding with agonist occupied and phosphorylated b2V2R by coimmunoprecipitation assay (Fig. 2b). We then tested b2V2Rbarr1-Fab30 complex by uorescence spectroscopy and interestingly found that incubation of actb2V2Rphos with mBBr-labelled barr1 indeed resulted in a decrease in uorescence intensity while that of Inactb2V2Rphos does not (Fig. 2c,d). Considering that both Inactb2V2Rphos and

Actb2V2Rphos interact with barr1 comparably, this observation suggest that the complexes of barr1 with Inactb2V2Rphos and actb2V2Rphos in fact represent, partially engaged (tail only) and fully engaged (tail core) complexes, respectively.

In order to further conrm this, we used an alternative approach where we rst assembled a complex of Apob2V2Rphos with barr1 and then incubated it with either an inverse agonist or agonist to generate partially and fully engaged complexes, respectively. We reasoned that Apob2V2Rphos should form a complex with barr1 primarily driven through the phosphorylated tail but it might also engage some core interaction owing to the constitutive activity of the receptor (Fig. 3a). We anticipated that incubation of this complex with inverse agonist should destabilize (and presumably ablate) the core interaction while agonist should further stabilize the core interaction. As presented in Fig. 3b,c,

Apob2V2Rphos indeed forms a stable complex with barr1, which is physically not altered by incubation with either the inverse agonist or agonist. Interestingly, however, bimane uorescence level in Apob2V2Rphosbarr1-Fab30 complex was lower compared with barr1( Fab30), suggesting a basal level of core engagement

in this complex (Fig. 3d,e). Incubation of this complex with agonist resulted in a robust decrease in uorescence intensity suggesting the engagement of core interaction. On the other hand, incubation with inverse agonist led to an increase in bimane uorescence bringing it up to barr1 alone level indicating disengagement of basal core interaction (Fig. 3d,e).

In order to further corroborate that bimane uorescence quenching is a reliable read out of core interaction, we tested a panel of receptor ligands with different efcacies on preformed

Apob2V2Rphos complex. Again, incubation of pre-formed complex with these ligands does not alter the physical interaction as assessed

IP: (Fab30) Prot. L

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Figure 2 | Validation of partially and fully engaged complexes by uorescence spectroscopy. (a) Structural model of b2ARb-arr1 complex deduced based on negative-stain electron microscopy, cross-linking experiments and hydrogen-deuterium exchange mass-spectrometry reveals nger loop of barr1 as a key component of the core interaction. L68 in the nger loop of barr1 was changed to cysteine in a cysteine-less barr1 and monobromobimane was attached to this cysteine by chemical coupling. Upon core interaction, bimane uorescence intensity decreases either due to change in chemical environment or quenching by a tyrosine/tryptophan residue in the vicinity. (b) Functional validation of bimane labelled barr1 by its interaction with puried b2V2R. Similar to wild-type barr1, bimane labelled barr1 also forms a complex with agonist occupied and phosphorylated b2V2R. The experiment was repeated twice with identical results and a representative image is shown. (c) Incubation of Actb2V2Rphos but not Inactb2V2Rphos with bimane labelled barr1 leads to a decrease in bimane uorescence. Considering equivalent physical interaction of Actb2V2Rphos and Inactb2V2Rphos (as presented in Fig. 1e,f), bimane uorescence data suggests that Actb2V2Rphos engages the core interaction while the Inactb2V2Rphos does not. These data suggest that

Inactb2V2Rphos barr1 Fab30 and Actb2V2Rphos barr1 Fab30 complexes represent tail only and fully (tail core) engaged complexes, respectively.

(d) Bimane uorescence at emission lmax as measured in c is presented as a bar graph. Data presented in d represent mean s.e.m. of three independent experiments analysed using one-way ANOVA with Bonferroni post-test (***Po0.001).

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Figure 3 | Ligand-dependent modulation of core interaction in Apob2V2Rphosbarr1-Fab30 complex. (a) A schematic representation showing that

Apob2V2Rphos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a tail only complex while incubation with an agonist stabilizes the core interaction and results in a fully engaged complex. (b) In-vitro assembly of Apob2V2Rphos complex with barr1 in presence of Fab30 as assessed by ELISA approach.

Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. (c) In-vitro assembly of

Apob2V2Rphos complex with barr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. (d) Incubation of pre-formed Apob2V2Rphos complex with inverse agonist (carazolol) results in an increase in bimane uorescence suggesting a loss of core binding, yet presumably stabilization of a tail engaged complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane uorescence suggesting the engagement of receptor core and, therefore, stabilization of a fully engaged complex. (e) Bimane uorescence at emission lmax as measured in d is presented as a bar graph. (f) Incubation of pre-formed Apob2V2Rphos complex with a panel of ligands results in different extent of bimane uorescence quenching, which directly correlates to the ligand efcacy.

(g) Quantication of decrease in bimane uorescence at emission lmax as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b, e and g represent means.e.m. of three independent experiments and analysed using one-way

ANOVA with Bonferroni post-test (*Po0.05; ***Po0.001).

by coimmunoprecipitation and enzyme-linked immunosorbent assay (ELISA) (Supplementary Fig. 4AC). Strikingly, however, the degree of uorescence quenching directly mirrors the ligand efcacy for the receptor (Fig. 3f,g). Furthermore, incubation of pre-formed complex with varying doses of the agonist (BI-167107) reveals that degree of uorescence quenching directly corresponds to the ligand occupancy of the receptor (Supplementary Fig. 4D).

These observations taken together with data presented in Fig. 2c,d conrm that the complexes of Inactb2V2Rphos and Actb2V2Rphos with barr1 represent, partially engaged (tail only) and fully engaged (tail core) complexes, respectively. It is interesting to

note here that we observe a decrease in bimane uorescence but not a shift in emission lmax. This indicates that the decrease in bimane uorescence most likely arises from quenching by a tyrosine or a

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tryptophan residue on the receptor and not directly from a different environment sensed by the bimane uorophore22.

Core interaction is dispensable for ERK2 binding. Activation of ERK MAP kinase has been extensively used as a primary readout of barr-dependent signalling downstream of GPCRs2326. barrs directly interact with ERK2 as well as upstream kinases of ERK cascade (c-Raf1 and MEK1) and it is proposed that barrs act as scaffolds to bring the components of ERK cascade together2730. We rst measured the interaction of puried barr1 with inactive and active ERK2 in the absence or presence of a phosphopeptide corresponding to the carboxyl terminus of the vasopressin receptor (V2Rpp). This phosphopeptide mimics the interaction of phosphorylated receptor tail and induces activation of barrs3133. We observed that barr1 interacts efciently with

ERK2/pERK2 and this interaction is not altered signicantly in the presence of V2Rpp (Supplementary Fig. 5). This nding suggests that activation of barr per se may not be required for its interaction with ERK2 and it prompted us to hypothesize that both, partially and fully engaged complexes should be able to interact with ERK efciently. Therefore, in order to test the functional competence of the partially engaged complex, we compared the binding of puried inactive and active ERK2 with fully engaged and partially engaged b2V2R-barr1ScFv30 complexes by ELISA and coimmunoprecipitation (Fig. 4ac and Supplementary Fig. 6). Here we used an ScFv variant of Fab 30, referred to as ScFv30 (Supplementary Fig. 6A), to stabilize the b2V2Rbarr1 complex in order to minimize any potential clash with ERK binding. Similar to Fab30, ScFv30 also effectively stabilizes b2V2Rbarr1 complex (Supplementary Fig. 6B).

Interestingly, as presented in Fig. 4b,c (and Supplementary Fig. 6DG), both inactb2V2Rphosbarr1 complex (tail engaged)

and actb2V2Rphosb-arr1 complex (fully engaged) exhibited robust binding to inactive (non-phosphorylated) and active (phosphorylated) ERK2. These data directly suggest that the core interaction in b2V2Rbarr1 complex is dispensable for

ERK binding. We note that the interaction of ERK2 MAP kinase with actb2V2Rphosbarr1 complex is slightly higher than inactb2V2Rphosbarr1 complex in the ELISA format and this observation perhaps reects relatively higher stability of the agonist bound quaternary complex under the experimental conditions.

In order to further corroborate these ndings, we utilized a previously described nanobody (referred to as Nb6B9) that selectively recognizes agonist bound b2AR conformation and represents a G protein mimetic34. CDR3 of this nanobody displays a signicantly overlapping interface on the receptor with that of the nger loop of barr1 (Fig. 4d). Therefore, we reasoned that pre-incubation of this nanobody with Actb2V2Rphos should preclude the nger loop mediated core interaction with barr1. We rst conrmed that binding of Nb6B9 to b2V2R does not affect the assembly of b2V2Rbarr1-Fab30 complex (Fig. 4e). We then tested the effect of Nb6B9 on bimane uorescence in b2V2Rbarr1-Fab30 complex. As presented in Fig. 4f, indeed pre-incubation of this nanobody to the receptor followed by addition of barr1 and Fab30 abolished bimane uorescence quenching that is observed in the absence of this nanobody. This data suggests that Nb6B9 blocks the core interaction between the b2V2R and barr1. Interestingly, however, the presence of this nanobody does not affect the interaction of the complex with active and inactive ERK2 MAP kinase (Fig. 4g,h). This observation taken together with the data presented in Fig. 4b,c conrms that the core interaction in b2V2Rbarr1 complex is dispensable for ERK binding.

Core interaction is dispensable for ERK activation. We next tested whether b2V2R engaged to barr1 only through the tail interaction is sufcient to trigger ERK activation in cells. As mentioned earlier, chemical cross-linking and structural modelling has identied the third intracellular loop in b2V2R as a major site for the core interaction with barr1 (Fig. 5a). In particular, Lys235 on the third intracellular loop of b2V2R cross-links with Lys77 in the nger loop of barr1 (Fig. 5a, inset). Furthermore, cross-linking studies and recent crystal structure of rhodopsin-visual arrestin complex has also identied the third intracellular loop as a part of the interface for the core interaction (Fig. 5b). Therefore, we generated a truncated b2V2R construct that harbours deletion of the third intracellular loop (D239 267;

referred to as b2V2RDICL3) (Fig. 5c). Agonist stimulation of HEK-293 cells expressing b2V2RDICL3 leads to signicant recruitment of barr1, albeit somewhat weaker than b2V2R, as assessed by confocal microscopy (Fig. 5d) and coimmuno-precipitation experiment (Fig. 5e). This data suggest that the absence of the third intracellular loop and, therefore, the core interaction does not ablate barr1 binding to the activated receptor in cellular context. In order to further conrm the interaction of b2V2RDICL3 with barr1 and the status of core interaction in its complex with barr1, we expressed and puried b2V2RDICL3 using baculovirus infected Sf9 cells (Supplementary Fig. 3). As presented in Fig. 5fh, puried b2V2RDICL3 formed a stable complex with barr1 in the presence of Fab30 as evaluated by ELISA and coimmunoprecipitation experiments. Most interestingly, b2V2RDICL3 even in the presence of agonist (that is, Actb2V2RDICL3-phos) did not exhibit any bimane uorescence quenching upon interaction with barr1 (Fig. 5i,j), indicating the inability of b2V2RDICL3 to engage the core interaction with barr1.

In order to further conrm the dispensability of the core interaction for ERK recruitment, we probed whether a complex of b2V2RDICL3 with barr1 can bind puried pERK2. As presented in

Fig. 6a and Supplementary Fig. 7A, b2V2RDICL3barr1-ScFv30 complex robustly recruited pERK2 and the level of interaction was comparable to that with analogous b2V2R complex. More importantly, stimulation of cells expressing b2V2RDICL3 with agonist isoproterenol leads to robust ERK activation similar to b2V2R (Fig. 6b,c). Of particular interest is the ERK activation at late time points (10, 20 and 30 min), which are well established to be mediated by barr-dependent and G protein independent pathway. These observations taken together with the data presented in Fig. 4 suggest that the core interaction in b2V2Rbarr1 complex is dispensable for ERK binding and activation. As mentioned earlier, the chimeric b2V2R behaves like a class B receptor with respect to barr interaction. Therefore, in order to probe whether the core interaction might be dispensable for class A receptors as well, we generated a native b2AR construct with truncated third intracellular loop, referred to as b2ARDICL3, and measured agonist induced ERK activation. Interestingly, we found that similar to b2V2R, truncation of the third intracellular loop in native b2AR also does not adversely affect ERK activation (Fig. 6d), suggesting that even for class A receptors, the core interaction may not be essential for stimulating ERK response.

In addition to ERK MAP kinase signalling, another key function of barrs is to promote GPCR internalization via clathrin coated machinery3537. It has been documented earlier that activation of barrs with isolated V2Rpp leads to robust clathrin binding32,33. In fact, as presented in Fig. 5d, confocal microscopy of cells expressing b2V2RDICL3 revealed that the truncated receptor is capable of internalization as reected by punctate appearance of barr1-YFP upon agonist stimulation. In order to further conrm whether core interaction is dispensable

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for receptor internalization as well, we rst measured the interaction of puried clathrin with partially and fully engaged complexes and observed comparable interaction (Supplementary

Fig. 7B). In addition, we also directly compared agonist-induced internalization of b2V2R and b2V2RDICL3 by measuring surface levels of the receptor in cells. As presented in Fig. 6e, b2V2RDICL3

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+ + +

+ + + +

2V2R

arr 1 ScFv30 p-ERK2

+ + +

+ + + +

d e

***

140

120

100

2AR

Absorbance 450 nm

(% normalized)

CDR3

***

Finger loop

Nanobody

2V2R

arr1 Fab30

+ +

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arr 1

f g h

*** ***

120

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arr1 + Fab30

arr1 + Fab 30 + Nb

arr1 + 2V2R + Fab 30 + Nb

arr1 + 2V2R + Fab30

140

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Fluorescence intensity at

max(normalized %)

Absorbance 450 nm

(% normalized)

Fluorescence intensity

(normalized %)

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Absorbance 450 nm

(% normalized)

2V2R

arr1 ScFv30

ERK2

Nb6B9 + Nb6B9

+ + +

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+ + +

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2V2R

arr1 ScFv30 pERK2

Nb6B9 + Nb6B9

+ + +

+ + + +

+ + +

+ + + +

0 400 440 480 520 560 600

***

Wavelength (nm)

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labelled barr1 did not result in any detectable quenching of bimane uorescence (Fig. 8f). These ndings indicate that in response to a barr biased ligand, receptor and barr might engage only through the phosphorylated carboxyl terminus without any signicant involvement of the core interaction.

DiscussionAgonist activation results in a conformational change in GPCRs which in turn leads to heterotrimeric G protein coupling and downstream responses. Activated receptors are phosphorylated by GRKs which then promotes the recruitment of barrs. It is generally believed that binding of barrs to GPCRs sterically precludes further G protein coupling leading to receptor desensitization44,45. In fact, superimposition of b2ARG protein complex crystal structure46 with electron microscopy based architecture of b2ARbarr1 complex11 reveals a signicantly overlapping interface on the receptor for barr1 and the Gas (Supplementary Fig. 8A). Moreover, crystal structure of rhodopsin with Ga C terminus peptide (GaCT)47 and arrestin nger loop peptide9 has revealed overlapping binding sites for the G protein and arrestin on the intracellular surface of the receptor.These observations indeed support steric hindrance based desensitization mechanism through competition for an overlapping interface on the cytoplasmic surface of the receptor. Interestingly, negative stain EM analysis of the b2V2Rbarr1 complex revealed a stable intermediate state in the biphasic interaction that represents a complex between b2V2R and barr1 associated solely through the phosphorylated carboxyl terminus of the receptor11. Stable isolation and direct visualization of this partially engaged complex underscores the sufciency of phosphorylated receptor tail for a physical complex formation with barr and hints at its potential functional signicance. Interestingly, crystal structure of pre-activated visual arrestin48 and V2Rpp bound barr131 have revealed major conformational changes compared with basal arrestin conformation. These changes include B20 movements of the

N- and the C-domain relative to each other and disruption of the polar core. These observations suggest that even partially engaged arrestin might be primed and conformationally competent to initiate at least some of barr functions. Our data presented here indeed suggest that partially engaged b2V2Rbarr1 complex associated only through the carboxyl terminus is sufcient to bind both, inactive and active ERK2. Furthermore, a truncated b2V2R lacking the 3rd intracellular loop and thereby defective in making core interaction with barr not only recruits barr1 in cells but also results in agonist stimulated ERK activation and receptor internalization. Considering these ndings, it is tempting to suggest that the core interaction between the GPCR and barrs might be essential for desensitization through steric hindrance while the tail interaction is sufcient, at least for some

Figure 4 | Core interaction is dispensable for recruitment of ERK2 MAP kinase. (a) An ELISA based approach to test the interaction of puried ERK2 with pre-formed b2V2Rbarr1-ScFv30 complex. Puried ERK2 (inactive or active) is immobilized on polystyrene surface followed by incubation with either the tail only engaged or fully engaged pre-formed complex. Interaction of ERK with the complex is visualized using HRP-coupled anti-FLAG M2 antibody as a read out of b2V2R retention on the plate. (b) Both tail only engaged (Inactb2V2Rphos b-arr1 ScFv30) and fully engaged (Actb2V2Rphos b-arr1 ScFv30)

complexes interact with immobilized inactive (non-phosphorylated) ERK2. (c) Similar to inactive ERK2, phosphorylated ERK2 (that is, active) also interacts with both, the tail only engaged and fully engaged complexes. (d) A previously described conformationally selective nanobody (Nb6B9) against agonist bound b2AR conformation has an overlapping interface with the core interaction. Structural representation based on superimposition of crystal structure of agonist bound b2AR and nanobody Nb6B9 (PDB ID:4LDO) and electron microscopy based model of b2V2Rbarr1 complex. (e) Pre-incubation of Actb2V2Rphos with puried Nb6B9 does not affect its physical interaction with barr1. Puried Actb2V2Rphos was rst incubated with a threefold molar excess of Nb6B9 and subsequently used for the assembly of b2V2Rbarr1-Fab30 complex in ELISA format. (f) Pre-incubation of Actb2V2Rphos with Nb6B9 abolishes bimane uorescence quenching observed upon interaction with barr1 suggesting that presence of Nb6B9 in Actb2ARphos barr1 Fab30 complex converts it to tail

only engaged complex. (g) Interaction of inactive ERK2 and (h) active ERK2 with Nb6B9 stabilized tail only engaged complex as assessed by ELISA, further suggests that the core interaction is dispensable for ERK recruitment. Data presented in b, c, e, g and h represent means.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test ( **Po0.01; ***Po0.001).

exhibits robust internalization upon agonist stimulation, even with slightly faster kinetics than b2V2R. Again, similar to ERK activation, we observed that b2ARDICL3 also undergoes robust endocytosis upon agonist stimulation (Fig. 6f). Taken together with the bimane uorescence data, this observation suggest that both, ERK activation and receptor internalization can be efciently supported by tail only engaged receptorbarr complex in the absence of core interaction.

There is some evidence in the literature that the second intracellular loop, R of DRY motif in particular, of GPCRs might also contribute to receptorbarr interaction38,39. Therefore, in order to test if ablating the potential contributions of the second intracellular loop towards the core interaction inuences barr recruitment and signalling, we inserted T4 lysozyme in the second intracellular loop of the b2V2R (between Lys141 and

Tyr142; construct referred to as b2V2R-T4LICL2) (Fig. 7ad). We reasoned that the bulky T4 lysozyme would separate the receptor core from barr through steric hindrance while not affecting barr1 recruitment through the phosphorylated tail. We also tested in parallel b2V2R constructs with T4L in the rst intracellular loop (T4L inserted between Gln65 and Thr66; b2V2R-T4LICL1) and third intracellular loop (T4L inserted between Glu238 and Glu268 with deletion of 239-267; b2V2R-T4LICL3) (Fig. 7ad). As presented in Fig. 7e, all these constructs exhibited barr1 recruitment to the receptor upon agonist stimulation as evaluated by confocal microscopy. More interestingly, these constructs also supported agonist induced ERK activation in cells similar to b2V2R and, therefore, indicate that the lack of potential contributions of rst and second intracellular loops towards core interaction can also be tolerated for ERK activation.

A b-arrestin biased ligand does not promote core interaction. An interesting avenue in GPCR signalling that has emerged recently is the concept of biased agonism40,41 and for several GPCRs, biased ligands are described that selectively trigger one or the other signalling pathways downstream of the receptor42. For perfectly biased barr biased ligands, there is no coupling of heterotrimeric G proteins and, therefore, no requirement of steric hindrance based desensitization of G protein signalling. We, therefore, hypothesized that a barr biased ligand may not promote core engagement between the receptor and barr. Carvedilol has been described as a high-afnity barr biased ligand for b2AR and it promotes barr interaction and ERK activation in the absence of any detectable G protein coupling43 (Fig. 8a). Carvedilol occupied b2V2R (referred to as Biasb2V2Rphos) exhibited a robust interaction with barr1 as assessed by ELISA (Fig. 8b) and coimmunoprecipitation (Fig. 8c). Furthermore, Biasb2V2Rphosbarr1-Fab30 complex also displayed robust interaction with inactive and active ERK (Fig. 8d,e). Most interestingly, the interaction of Biasb2V2Rphos with bimane

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of the functional outcomes such as ERK binding, activation and receptor internalization (Supplementary Fig. 8B).

Based on their relative patterns of barr recruitment, GPCRs are broadly categorized as either class A or class B receptors18. Class A receptors, such as b2AR, bind transiently to barrs and show rapid recycling to the cell surface after internalization. Class B receptors on the other hand, such as V2R, exhibit a more robust

interaction with barrs and show proteosomal degradation. Class B receptors typically harbour phosphorylatable Ser/Thr clusters in their carboxyl terminus while class A receptors appear to primarily have more scattered Ser/Thr residues. It is conceivable that such clusters of Ser/Thr in class B receptors impart a stronger cumulative contribution towards higher afnity for barrs. Two recent studies using FlAsH based barr2 sensors suggest distinct

a b

Finger loop

2AR

TM5 TM6

Rhodopsin

TM5

Ser240

Asp83

TM6

K235

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ICL3

K77

arr1

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280

2V2RICL3

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arr 1Fab30

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ICL3-phos

Inact 2 V 2 R

Act 2 V 2 R

Inact 2 V 2 R

Act 2 V 2 R

Act2V2RICL3-phos

120 +arr1+Fab30 Inact2V2RICL3-phos +arr1+Fab30

arr1+Fab30

Signal intensity

(% normalized)

Act 2 V 2 R

Inact 2 V 2 R

WB: FLAG (2V2RICL3)

WB: Prot. L (Fab30)

IP: Prot. L (Fab30)

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(% normalized)

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at [afii9838] max(% normalized)

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arr 1

+ +

arr 1

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0 400 440 480 520 560 600

40 arr 1+Fab30

Wavelength (nm)

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probe such a scenario where differently engaged GPCRbarr complexes carry out different sub-sets of functions and this might help establish a mechanistic basis for broad functional repertoire and effective functional segregation along the GPCR-barr signalling axis. It should also be noted that even with ICL3 truncated chimeric b2V2R or with other class B GPCRs, some transient core interaction can still occur, which escapes detection in bimane uorescence assay but might still contribute towards some of the functional outcomes.

The concept of biased GPCR signalling and development of biased ligands has rened the general understanding of receptor pharmacology6163. For many GPCRs, biased ligands are proposed to represent better therapeutic potential over currently prescribed ones by virtue of having reduced side effects42. However, the mechanistic and structural insights into biased GPCR signalling remains relatively less well dened. It is proposed that biased ligands induce a distinct set of conformations in the receptor than unbiased ligands and these different conformations are subsequently recognized by downstream effectors such as barrs11,64. As a result, effectors also adopt distinct conformations which in turn govern their functional outcome50,65. A recent study using unnatural amino acid incorporation and 19F-NMR on barr1 has investigated the connection between barr1 conformation and functional outcome66. This study suggests that different phosphopeptides harbouring differential phosphorylation patterns that potentially correspond to a bar-code imparted by different GRKs are capable of inducing distinct conformations in barr1.

These distinct conformations in turn ne-tune the functional outcome of barr1 such as clathrin binding and c-Src activation66.

Furthermore, two recent reports using barr2 conformational sensors also suggest that not only different receptors impose different conformational signature on barr2, but also ligands of different efcacies (such as unbiased and biased) induce detectably different conformations in barr249,50. However, it currently remains unknown whether a GPCRbarr complex in response to a biased ligand is conformationally and structurally different than that in response to unbiased ligand. As barr biased ligands selectively trigger barr recruitment in the absence of any G protein activation, there is no requirement of desensitization of G protein signalling. Therefore, it is logical to speculate that barr may not be required to fully engage with the receptor core. Our ndings that carvedilol, a barr biased b2AR ligand, does not engage core interaction between the receptor and barr1 in fact supports such a possibility. Although carvedilol has a weak efcacy for barr-dependent b2AR signalling, 19F NMR based analysis of carvedilol bound b2AR67 as well as chemical labelling approach68 has directly demonstrated that it promotes distinct conformational changes in

Figure 5 | Truncation of the third intracellular loop in b2V2R ablates core interaction with barr1. (a) Cross-linking experiments and electron microscopy based structural model of b2V2Rbarr1 complex has identied the third intracellular loop of the b2V2R as prominent interface for core interaction through docking of the nger loop of barr1. Residues that are identied to cross-link with each other in b2V2Rbarr1 complex are labelled and their side chains are highlighted as space ll model. (b) Cross-linking studies and X-ray crystal structure of rhodopsin-visual arrestin also displays the vicinity of the third intracellular loop in rhodopsin with the nger loop of visual arrestin. (c) Sequence alignment of b2V2R and b2V2RDICL3 (third intracellular loop truncated receptor) to highlight the deleted amino acids (Gly238-Lys267) (red box). (d) Confocal microscopy of HEK-293 cells expressing either b2V2R or b2V2RDICL3 with b-arr1-YFP. Agonist stimulation leads to accumulation of endocytotic vesicles that indicates recruitment of barr1 to activated receptor. Nuclear staining is shown using 4,6-diamidino-2-phenylindole. Compared with b2V2R, b2V2RDICL3 exhibits somewhat weaker recruitment of barr1 as reected by less punctate appearance. Scale bar, 10 mm. (e) Coimmunoprecipitation of b2V2RDICL3 with barr1 expressed in HEK-293 cells further conrms the recruitment of barr1 to the truncated receptor upon agonist stimulation. Cells were stimulated with agonist (Isoproterenol, 10 mM for 30 min at 37 C) followed by cross-linking using dithiobis(succinimidyl-propionate) (1 mM for 30 min at room-temperature) and subsequently, receptorbarr1 complex was coimmuno-precipitation using anti-FLAG antibody beads. (f) Assembly of b2V2RDICL3 b-arr1 Fab30 complex as measured using ELISA approach and (g)

coimmunoprecipitation experiment. Similar to b2V2R, b2V2RDICL3 also forms a stable complex with barr1 in the presence of Fab30. (h) Quantication of b2V2RDICL3barr1 complex formation as assessed by coimmunoprecipitation. (i) Bimane uorescence spectroscopy on b2V2RDICL3 complex reveals the absence of uorescence quenching even in the presence of agonist and thereby suggests the lack of core interaction. (j) Bimane uorescence at emission lmax as measured in i is presented as bar graph. Data in f represents means.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (***Po0.001). Data in g and h represent two independent experiments.

conformational signatures of barr2 imparted by class A vs class B GPCRs49,50. Although we have primarily used a chimeric receptor, b2V2R that displays class B prole of barr recruitment, we also demonstrate that even for a prototypical class A GPCR, b2AR, core interaction is not essential for ERK activation and internalization. This observation indicates that both, class A and B receptors are capable of undergoing endocytosis and triggering ERK activation when engaged with barrs only through the phosphorylated carboxyl terminus. Along similar lines, a recent investigation has documented that barr2 can mediate ERK activation downstream of b1AR despite a very transient interaction and dissociation from the receptor51,52.

Going forward, it would be interesting to test additional receptor systems to evaluate the generality of these observations in a broader context.

Constitutive activity of GPCRs refers to the basal level of activation even in the absence of activating ligand. For a number of GPCRs, constitutive activity has been detected with respect to G protein activation and it is thought to arise from the abilities of the receptors to sample active like conformations even in the absence of activating ligands. Here we observe that there is a small but signicant core interaction between the Apo-receptor and barr as assed by bimane uorescence spectroscopy (Fig. 3), which is destabilized or stabilized by the incubation of this complex with inverse agonists or agonists, respectively. These ndings raise the possibility that some basal level of barr recruitment might exist in cells even in the absence of stimulating ligand and in fact may be responsible for desensitizing the constitutive receptor activity and some basal level of barr signalling. Future investigations will be required to carefully probe this aspect of GPCR signalling.

It is important to mention that barrs mediate and regulate multiple functions downstream of GPCRs. For example, barrs can scaffold the components of clathrin mediate internalization machinery such as clathrin and AP2 and have a key role in GPCR internalization35,53. In addition to ERK MAP kinase, barrs also scaffold components of other MAP kinase pathways (such as JNK54,55, p38) as well as c-Src56 and Akt57. Furthermore, scaffolding of E3 ubiquitin ligases has also emerged as a new functional role of barrs for GPCRs and non-GPCR membrane proteins5860. Although our data suggest that barr1 engaged to the receptor only through the phosphorylated carboxyl terminus is competent to recruit and activate ERK MAP kinase and support receptor internalization, it is plausible that core interaction might still be required for some of the other functional aspects of GPCRbarr complex, in addition to receptor desensitization. Further investigations are required to

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ICL3-phos

a b

ICL3-phos

Inact- 2 V 2 R

Act- 2 V 2 R

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Isoproterenol (min)

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Figure 6 | Truncation of the third intracellular loop does affect ERK activation and internalization. (a) Interaction of phosphorylated ERK2 MAP Kinase with b2V2RDICL3 barr1 ScFv30 complex as assessed by ELISA approach. Similar to b2V2R, b2V2RDICL3 also forms stable complexes with phosphorylated

ERK2. Data represent means.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (***Po0.001). (b) Agonist induced activation of ERK1/2 MAP kinase for b2V2R and b2V2RDICL3 shows a similar temporal pattern suggesting that truncation of the third intracellular loop, and, therefore, ablation of the core interaction, does not signicantly affect ERK activation. The experiment was repeated four times with identical results and a representative image is shown. (c) Quantication of the ERK activation data presented as means.e.m. of four independent experiments. (d) Agonist induced activation of ERK1/2 MAP kinase downstream of b2ARWT and b2ARDICL3 also reveals similar pattern suggesting the dispensability of the core interaction even for class A receptors. A representative image and quantitation of seven independent experiments are shown. (e) Similar to ERK activation, agonist induced internalization of b2V2RDICL3 also exhibits a comparable pattern to b2V2RWT albeit with an increased kinetics. (f) b2ARDICL3 also undergoes robust internalization upon agonist stimulation similar to b2ARWT. Data in e and f represent six independent experiments each carried out in duplicate and presented as means.e.m.

the receptor compared with unbiased agonists or inverse agonists. However, further experimentation with other GPCRs that have more efcacious biased ligand is desirable to probe the generalization of this observation.

In conclusion, our ndings reveal a previously unknown aspect of GPCRbarr interaction and provide a potential basis for broad functional repertoire of this signalling axis. In contrast with generally anticipated notion, we demonstrate that partially

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engaged GPCRbarr complex is functionally competent with respect to supporting receptor internalization, and recruitment and activation of ERK MAP Kinase. Our data also suggest that barr biased ligands may not engage the receptor core with barr and, therefore, identify a key mechanistic insight in to biased agonism. It would be very interesting to investigate in future

whether other conserved barr functions might also be carried out through partial engagement with the activated GPCRs.

Methods

General reagents and protein expression. General chemicals and cellculture consumables were purchased from Sigma-Aldrich or local vendors unless specied otherwise. Codon optimized barr1 gene was synthesized (Genscript), sub-cloned in to pGEX4T3 vector (purchased from GE), expressed inE. coli (BL21) and puried using Glutathione Sepharose afnity resin33. Codon optimized Fab30 open reading frame was synthesized (Genscript) based on published crystal structure (PDB ID: 4JQI) (ref. 31), expressed and puried in M55244 strain of E. coli (purchased from American Type Culture Collection)69. As an alternative strategy, the coding regions for the light and heavy chains of Fab30 were cloned in pETDuet-1 vector (Novagen), expressed in BL21 (DE3) cells (NEB) with 0.5 mM isopropyl-b-D-thiogalactoside induction at 18 C for 1216 h (Supplementary Fig. 1). Subsequently, Fab30 was puried from total lysate on Protein L resin (purchased from GE)69. The coding region of nanobody Nb6B9 was synthesized based on previously published crystal structure (PDB ID: 4LDO) (ref. 34) and it was expressed in E. coli (Rosetta) (NEB) and puried using Ni-NTA afnity chromatography34. Coding region of barr1-Cys68 was synthesized (Genscript) and cloned in pGEX4T3 vector followed by expression in E. coli (BL21) and purication on Glutathione Sepharose afnity resin (Clonetech).

Coding region of human ERK2 and constitutively active MEK1 (R4F) were synthesized (Genscript), cloned in pGEX4T3 vector and expressed in E.coli ShufeT7 express cells (NEB). Protein expression was induced at OD600 0.60.8 with 0.2 mM isopropyl-b-D-thiogalactoside at 16 C for 1216 h. Cell pellets were resuspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethyl sulphonyl uoride, 0.25 mM dithiothreitol and lysozyme for 1 h at 4 C. Cell suspension was sonicated, centrifuged and then loaded on to a preequilibrated Glutathione-Sepharose resin (GE). After overnight binding at 4 C, beads were washed extensively and then proteins were eluted using thrombin protease (Sigma or Merck). For ERK2 phosphorylation, a reaction containing inactive ERK2 and constitutive active MEK1 (R4F) in phosphorylation buffer(20 mM HEPES, pH 7.0, 5 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 100200 nM ATP) was prepared and incubated for 1 h at 30 C. The reaction was quenched by addition of stop buffer (50 mM Tris pH 7.5, 18 mM EDTA), followed by a buffer exchange step on a PD10 column. Phosphorylation of ERK2 was validated by western blotting with phospho-ERK antibody (CST, catalog number. 9101; 1:5,000 dilution).

Open reading frames of FLAG-b2V2R chimeric receptor and GRK2CAAX were synthesized (Genscript) and baculovirus stocks were generated using standard protocols (Expression Systems). FLAG-b2V2R and GRK2CAAX were co-expressed in Sf9 cells (purchased from Expression Systems) and cultured in ESF921 media (Expression Systems) and 6066 h post-infection; cells were stimulated with indicated ligand, harvested and lysed by glass douncing. Subsequently, cells were solubilized using 0.5% (w/v) maltose neopentyl glycol (MNG, purchased from Anatrace) and puried on anti-FLAG M1 afnity resin (Sigma). Puried protein samples were either used fresh in the experiments or ash-frozen in small aliquots after addition of 1020% glycerol and stored at 80 C until further use.

ELISA based assembly of b2V2Rbarr1-Fab30 complex. For ELISA based in-vitro assembly of b2V2Rbarr1Fab/ScFv30 complexes, puried Fab/ScFv30 (in 20 mM Hepes, pH 7.4, 100 mM NaCl) was rst immobilized on 96 well MaxiSorp polystyrene plates (Nunc) at room temperature for 1 h. Afterwards, potential non-specic binding sites in the wells were blocked by incubation with 1% BSA at room temperature for 1 h. Subsequently, mixture of ligand stimulated cell lysate (or puried receptor) was added to the wells and incubated at room temperature for 1 h. Wells were washed extensively using 20 mM Hepes, pH 7.4, 100 mM NaCl, 0.01% MNG and then incubated with 1:2,000 dilution of HRP-coupled anti-FLAG M2 antibody (Sigma, catalog number A8592). After 1 h incubation, wells were extensively washed and assembly of the complex was visualized by adding 3,30,5,50-tetramethylbenzidine (TMB) ELISA (Genscript or

Thermo). Colorimetric reaction was stopped by adding 1M H2SO4 and absorbance

a b

2V2R-T4LICL1

2V2R-T4LICL2

T4L

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2V 2R-T4L

Figure 7 | Blocking the potential contribution of intracellular loops does not affect ERK activation. Schematic illustration of b2V2R constructs with

T4 lysozyme insertion in (a) intracellular loop 1 between Gln65 and Thr66 (b) intracellular loop 2 between Lys141 and Tyr142and (c) intracellular loop 3 between Glu238 and Glu268 with deletion of 239-267. (d) Expression of b2V2R-T4L constructs in transfected HEK-293 cells as visualized by western blotting using N-terminal FLAG tag. (e) Agonist induced barr1 recruitment to b2V2R-T4L constructs as visualized by confocal microscopy in HEK-293 cells expressing barr1-YFP. Scale bar, 10 mm. (f) Agonist (Isoproterenol, 10 mM) induced ERK1/2 activation in HEK-293 cells expressing b2V2R-T4L constructs at indicated time points. Data inf show a representative image of three independent experiments.

Iso. (min)

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a b c

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Figure 8 | A barr biased ligand of b2AR does not promote core interaction with barr1. (a) Carvedilol is a high-afnity barr biased ligand of b2AR and it selectively promotes barr binding and ERK activation in the absence of any detectable G protein coupling. (b) Carvedilol bound and phosphorylated b2V2R

(referred to as Biasb2V2Rphosgenerated through incubation of Apob2V2Rphos with tenfold molar excess of carvedilol) exhibits a robust interaction with barr1 in the presence of Fab30 as assessed by ELISA. Puried Fab30 was immobilized and then incubated with barr1 and either Biasb2V2Rphos or Actb2V2Rphos.

Formation of complex was detected using anti-FLAG M2 antibody. (c) Formation of barr1 complex with Biasb2V2Rphos in the presence of Fab30 as assessed by coimmunoprecipitation. The experiment was repeated three times with identical results and a representative image is shown. Quantication of the data is shown as bar graph. (d) Interaction of Biasb2ARphos barr1 ScFv30 and Actb2V2Rphos barr1 ScFv30 complexes with inactive and

(e) active ERK2. Puried ERK2 was immobilized followed by incubation with pre-formed complexes and detection using HRP-coupled anti-FLAG M2 antibody. (f) Interaction of Biasb2V2Rphos with barr1 does not lead to a detectable decrease in bimane uorescence suggesting the lack of core interaction.

Apob2V2Rphos was rst incubated with tenfold molar excess of carvedilol or BI-167107 to obtain Biasb2V2Rphos and Actb2V2Rphos, respectively. Subsequently, these receptor preparations were incubated with bimane labelled barr1 and Fab 30 to form a complex followed by uorescence scanning in the wavelength range indicated on the graph. The data represent an average of three independent experiments. Data presented in b, d and e represent means.e.m. of at least three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferronipost-test (**Po0.01; ***Po0.001).

was measured at 450 nm using a Victor X4 plate reader (Perkin-Elmer). All the ELISA data are normalized with respect to the signal for Actb2V2Rphos complex which is treated as 100%.

For dephosphorylation experiment, cell lysate was incubated with lphosphatase (NEB) at 25 C for 2 h and subsequently used for in-vitro assembly of the complex. Fab CTL represents a random Fab taken from the library as a negative control. For dose response ELISA experiment, different amounts of b2V2Rbarr1

mixture were added to the Fab30 coated anchor surface followed by blocking of

non-specic binding surface and complex detection.

Bimane uorescence spectroscopy. Puried barr1L68C was buffer exchanged in 20 mM Hepes, 150 mM NaCl, pH 7.5 buffer and concentrated to B2.0 mg ml 1. It was incubated with 10-fold molar excess of monobromobimane (mBBr,

Sigma-Aldrich) on ice for 1 h. Subsequently, the sample was centrifuged at 100,000g for 30 min to remove aggregates and then unreacted mBBr was separated on a PD10 desalting column (GE Healthcare). Labelled protein was either used in bimane uorescence experiment right away or ash frozen with 20% glycerol for

later usage. Labelling efciency of barr1L68C under these conditions was measured to be about 85%. For uorescence experiments, mBBr labelled barr1L68C was used at an approximate nal concentration of 2 mM and it was mixed with threefold molar excess (6 mM) of puried b2V2R and Fab30 for 60 min at room temperature (25 C). For the experiments presented in Fig. 2 and Fig. 8, puried Apob2V2Rphos was pre-incubated with 510 fold molar excess (3060 mM) of respective ligands (30 min at 25 C) before mixing it with barr1 and Fab30. For the experiments presented in Fig. 3, the complex of Apob2V2Rphosbar1-Fab30 (6 mM:2 mM:2 mM) was allowed to form at 25 C followed by addition of 510 fold molar excess of ligand (3060 mM) and an additional 30 min incubation at 25 C. Fluorescence scanning analysis was performed using Fluorimeter (Perkin Elmer, USA model LS-55) in photon counting mode by setting the excitation and emission band pass lter of 5 nm. For emission scan, excitation was set at 397 nm and emission was measured from 415 nm to 600 nm with scan speed of 50 nm min 1. Bimane uorescence intensities in each experiment are normalized with respect tobarr1 Fab30 condition, which is treated as 100%. Fluorescence intensity was also

corrected for background uorescence from buffer and protein in all experiments and each experiment was repeated at least three times.

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ERK assay and confocal microscopy. HEK-293 cells (purchased from American Type Culture Collection) were cultured in Dulbeccos modied Eagles complete media (Sigma) supplemented with 10% fetal bovine serum (Thermo Scientic) and 1% penicillinstreptomycin at 37 C under 5% CO2. For protein expression, cells were transfected with indicated plasmids using PEI (Polyethylenimine) as the transfection reagent at a DNA to PEI ratio of 1:3 (7 mg of DNA mixed with 21 ml of

PEI). Cells were serum starved for 412 h and then stimulated with appropriate ligands as indicated in the gure legends.

For cross-linking of b2V2RDICL3 and barr1, Carazolol and BI-167107 stimulated HEK-293 cells were resuspended in buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, 1 PhosStop (Roche) and 1 complete protease inhibitor

(Sigma). Cells were lysed by dounce homogenization. For cross-linking, 1 mM dithiobis(succinimidyl-propionate) (Sigma) in dimethylsulphoxide was added from 100 mM stock and lysate was tumbled at room temperature for 30 min. The reaction was quenched by adding 1M Tris buffer pH 8.0 and 1% (v/v) MNG was added for solubilization and tumbled for 3 h at 4 C. Following solubilization, lysate was centrifuged at 21,130g for 30 min. The clear supernatant was collected in separate tube and freshly equilibrated M1 FLAG beads were added for immunoprecipitation. Coimmunoprecipitated barr and b2V2R were detected by western blotting rabbit mAb anti-barr antibody (CST, 1:1,000, catalog number D24H9) and HRP-coupled mouse anti-FLAG M2 mAb (Sigma, 1:1,000). Blots were developed on Chemidoc (Bio-Rad) and subsequently quantied by ImageLab software (Bio-Rad).

For ERK assay, transfected cells were seeded in to six-well plates (Corning), serum starved for 12 h and then stimulated with 10 mM Isoproterenol (Sigma-

Aldrich) for indicated time points. Subsequently, the cells were lysed in 200 ml of 2 SDS loading buffer, sonicated and loaded on to 12% SDSpolyacrylamide gel

electrophoresis. Western blotting was performed to observe the phosphorylation of ERK1/2. The bands were transferred on PVDF membrane (BioRad). The membrane was blocked with 5% BSA (SRL) for 1 h and then probed with anti-pERK primary antibody (CST, catalog number. 9101; 1:5,000 dilution) overnight at 4 C followed by 1 h incubation with anti-rabbit IgG secondary antibody (Genscript, catalog number. A00098) at room temperature. The membrane was then washed with 1 TBST thrice and developed using Chemi

Doc (BioRad). The anti-pERK antibody was stripped-off using 1X stripping buffer and then reprobed with anti-tERK antibody (CST, catalog number. 9102 and 4695; 1:5,000 dilution).

For confocal microscopy, transfected HEK-293 cells were seeded onto 0.001% poly-L-lysine coated glass coverslips and serum starved for 4 h. Cells were then stimulated with 10 mM Isoproterenol for indicated time points, xed using 4%

paraformaldehyde and permeabilized with 0.05% Triton-X-100. For nuclear staining, 0.5 mg ml 1 of 4,6-diamidino-2-phenylindole solution (Sigma) was added to xed cells. After nal washing with PBS, coverslips were mounted on to glass slides using VectaShield H-1,000 mounting medium (VectaShield), allowed to air dry for 15 min and then imaged using LSM780NLO confocal microscope(Carl Zeiss).

Coimmunoprecipitation experiments. In order to assess the formation of b2V2Rbarr1 complex in solution by coimmunoprecipitation, puried b2V2R(2.5 mg) was mixed with puried barr1 (2.5 or 5 mg) and Fab30 (2.5 mg) and incubated at room-temperature for 1 h. Subsequently, 20 ml of protein L beads (Capto L, GE Healthcare) were added and the mixture was allowed to tumble at room-temperature for additional 1 h. Afterwards, beads were washed three times with washing buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 0.01% MNG) and eluted with SDS loading buffer. Eluted samples were separated by 12% SDS polyacrylamide gel electrophoresis and probed using HRP-coupled anti-FLAG M2 antibody (Sigma, 1:2,000) and HRP- coupled protein L (GenScript, 1:2,000; catalog number M00098) by western blotting.

In order to measure binding of ERK2 with pre-formed complex, puried GST-ERK2 (or GST-pERK2) (6 mg) was immobilized on freshly equilibrated

GS beads (1 h at room-temperature) and washed once with washing buffer to remove unbound GST-ERK2. Subsequently, beads were incubated (1 h at room-temperature) with pre-formed b2V2Rbarr1ScFv30 complex(4 mg:4 mg:5 mg) followed with three washes. Afterwards, bound samples were eluted in SDS loading buffer and probed by western blotting using HRP-coupled anti-FLAG M2 antibody. Puried GST was used as a control for non-specic binding of the complex to GS beads. Quantication of coIP data is normalized with respect to Actb2V2Rphos, which is treated as 100%.

Receptor internalization assay. HEK-293 cells expressing b2V2R and b2V2RDICL3 seeded in to 24-well plates at a density of 300,000 cells per well and serum starved for 2 h. Cells were stimulated with 10 mM isoproterenol at specied timepoints followed by three washes with ice cold tris-buffered saline (TBS) and subsequently xed with 4% (w/v) paraformaldehyde for 20 min on ice. Cells were again washed with TBS and blocked with TBS 1%(w/v) BSA for 1 h at room

temperature. Cells were then incubated with HRP-coupled anti-FLAG M2 antibody (Sigma) at a dilution of 1:1,000 in TBS 1%BSA for 1 h at room temperature.

Afterwards, cells were washed with TBS 1%(w/v) BSA three times and incubated

with 200 ml 3,30,5,50-tetramethylbenzidine (TMB) per well for visualizing surface receptor expression. Reaction was stopped by transferring 100 ml of developed

solution to a 96-well plate already containing 100 ml of 1M H2SO4. Plates were read at 450 nm in a microplate reader (Victor X4). For measuring total protein (for normalization), cells were washed with TBS and 200 ml of 0.2% (w/v) Janus green stain was added per well and incubated for 10 min. Subsequently, cells were destained with water until excess dye was removed and colour was developed by adding 800 ml of 0.5 M HCl per well. One-hundred microlitres of solution was transferred in 96-well plate and read at 595 nm in a multi-plate reader. The values were normalized by dividing A450 reading with A595 reading.

Data analysis. All the data were plotted using GraphPad Prism software and analysed as indicated in the gure legends. For statistical analysis, we used one-way ANOVA with Bonferroni post-test. Uncropped images of key experiments are presented in the Supplementary Fig. 9.

Data availability. The crystal structures of Fab30 and nanobody Nb6B9 bound to b-arrestin were obtained from PDB using accession codes 4JQI and 4LDO, respectively. The data that support the ndings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the members of Dr Shuklas laboratory for scintillating discussion and critical reading of the manuscript. The research program in Dr Shuklas laboratory is supported by the Indian Institute of Technology Kanpur (IITK /BSBE/2014011), the Department of Science and Technology (DST), Council of Scientic and Industrial Research (CSIR) and the Wellcome Trust/DBT India Alliance. Dr Shukla is an Intermediate Fellow of Wellcome Trust/DBT India Alliance (IA/I/14/1/501285). Synthesis of BI-167107 in Dr Xin Chens laboratory was supported by a grant from the National Science Foundation of China (No. 21272029). We thankfully acknowledgeDr Linton M. Traub, University of Pittsburg, for proving Clathrin-TD plasmid. We thank Mr Arvind Kumar for excellent secretarial assistance. We thank Kumari Nidhi for help in the early stages of ERK assay and Dr Mithu Baidya in b2AR endocytosis experiments. We also thank Prof. Ashwani K. Thakur for kindly allowing us to use the Fluorescence Spectrometer in his laboratory.

Author contributions

P.K. designed and carried out the ELISA and coIP experiments for complex assembly and ERK binding and performed ERK assay for b2AR; A.S. expressed and puried barr1L68C, functionally validated its interaction with b2V2R and carried out bimane uorescence experiments with help from R.B.; R.B. cloned, expressed and puried b2V2R (WT and

DICL3) with help from P.K. and assisted in bimane uorescence experiments; E.G. carried out the cross-linking coIP experiment to conrm the interaction of b2AV2RDICL3 with barr1 in cells and performed receptor internalization assays, X.C. provided BI-167107; P.G. carried out confocal microscopy with help from C.G. and ERK assay on b2V2R constructs with help from P.K.; R.R. expressed and puried Fab30 with help from

D.J., ScFv30 and Nb6B9; B.G. expressed and puried ERK2 and MEK1, and performed ERK2 phosphorylation. A.K.S. managed and supervised overall project. All authors contributed to data analysis, interpretation and writing of the manuscript.

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G Protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors and drug targets. GPCR signalling and desensitization is critically regulated by β-arrestins (βarr). GPCR-βarr interaction is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N-domain of βarr first and then seven transmembrane core of the receptor engages with βarr. It is currently unknown whether fully engaged GPCR-βarr complex is essential for functional outcomes or partially engaged complex can also be functionally competent. Here we assemble partially and fully engaged complexes of a chimeric β₂ V₂ R with βarr1, and discover that the core interaction is dispensable for receptor endocytosis, ERK MAP kinase binding and activation. Furthermore, we observe that carvedilol, a βarr biased ligand, does not promote detectable engagement between βarr1 and the receptor core. These findings uncover a previously unknown aspect of GPCR-βarr interaction and provide novel insights into GPCR signalling and regulatory paradigms.

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Title

Functional competence of a partially engaged GPCR-[beta]-arrestin complex

Author

Kumari, Punita; Srivastava, Ashish; Banerjee, Ramanuj; Ghosh, Eshan; Gupta, Pragya; Ranjan, Ravi; Chen, Xin; Gupta, Bhagyashri; Gupta, Charu; Jaiman, Deepika; Shukla, Arun K

Pages

13416

Publication year

2016

Publication date

Nov 2016

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms13416

ProQuest document ID

1837226742

Functional competence of a partially engaged GPCR-[beta]-arrestin complex

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