GPCR dimerization: Drug discovery aspects and targets in renin‐angiotensin systems

Abstract

G protein‐coupled receptors (GPCRs), which play crucial roles in various physiological functions, often assembled into dimers and higher‐order oligomers. This oligomerization phenomenon has been observed in diverse physiological and pathological contexts, presenting promising opportunities for drug discovery targeting vital systems such as the cardiovascular, nervous, endocrine, and renal systems. This review offers a concise understanding of GPCR dimerization, its signaling mechanisms, and its implications. Furthermore, we explored therapeutic strategies aimed at modulating receptors involved in dimer/oligomer formation within the renin‐angiotensin system.

INTRODUCTION

G protein-coupled receptors (GPCRs) constitute the largest receptor family, orchestrating signaling pathways vital for numerous physiological processes including sensation,¹ neural transmission, inflammation, cellular growth and differentiation, muscle contraction and relaxation, and sodium/fluid homeostasis.² Typically situated on the cell membrane, GPCRs feature seven transmembrane domains (7-TM) structured by three intracellular loops (ILs) and three extracellular loops (ELs), with an intracellular C-terminal and an extracellular N-terminal domain.³ Upon ligand binding, these transmembrane structures undergo conformational changes, facilitating diverse three-dimensional configurations of GPCRs. In addition to single receptor originated downstream signaling, GPCRs can interact with each other, to form dimers or oligomers that synchronize the signaling pathways. While it was initially believed that monomeric GPCRs solely interact with single heterotrimeric G proteins in response to external stimuli, further exploration has revealed a more intricate landscape. Although dimerization is evident in many GPCRs, the specific interactions leading to dimer formation and their significance in pharmacological signaling remain the subjects of ongoing investigation. Notably, a substantial percentage of drugs (~35%) target GPCRs, with over 134 small molecules or peptides approved for use in the USA and the European Union.⁴ Moreover, a considerable number of GPCRs (~128 out of approximately n 800 known GPCRs) lack known agonists (orphans), rendering them potential candidates for drug discovery efforts, particularly within the context of dimer formation.⁵ This review aims to elucidate the fundamental aspects of GPCR dimerization within a broader perspective of drug discovery. Additionally, within the renin-angiotensin system, GPCR dimers and oligomers, such as angiotensin receptors, Mas receptors, and bradykinin receptors, play a pivotal role in regulating blood pressure, fluid homeostasis, and the maintenance of electrolyte balance, underscoring their significance in physiological processes. Hence, these receptors have been specifically chosen for detailed examination.

MECHANISMS OF DIMERIZATION

In the typical mode of GPCR signal transduction, upon ligand binding, GPCR interacts with the Gαβγ heterotrimeric protein, facilitating the exchange of GTP with Gα subunit bound GDP. This exchange initiates dissociation of the Gα and Gβγ subunits, which then interact with their respective effector molecules. In humans, 16 genes have been identified to encode 21 Gα subunits that interact with diverse effector molecules.⁶ For instance, Gαs stimulates adenylate cyclase (AC) activity, leading to increased production of the second messenger cAMP, whereas Gαi inhibits AC, consequently decreasing cAMP levels⁷ Similarly, Gαq activates phospholipase Cβ (PLCβ), and Gα11 activates Bruton's tyrosine kinase.⁸ Initially, these interactions were thought to be random, following the “collision coupling model,”⁹ which aligns with the “fluid mosaic” model of the plasma membrane.^10,11 Evidence also suggests the existence of permanent pre-coupled GPCRs and G proteins, as observed in the adenosine 2A receptor. Recent studies have revealed receptor–receptor interactions and cross-talks, indicating their physiological and pharmacological significance in altering signal transduction pathways. This interaction between two identical receptors is termed as homo-dimerization, the interaction between two different receptors is hetero-dimerization and the involvement of more than two receptors is termed oligomerization. For example, Patel et al. (2017) demonstrated that AT2-Mas receptor heterodimer formation in the renin-angiotensin system is facilitated by the close proximity (<7Å) between these two 7-transmembrane receptors with formation of disulfide bridges, particularly under oxidative conditions.¹² Some GPCRs, which do not strictly follow G protein coupling, exhibit dimerization or oligomerization involving different transmembrane domains (e.g., TMD VI in β2 adrenoceptor, TMD IV and V in rhodopsin, TMD IV in dopamine) or extracellular domains (e.g., Bradykinin B2 receptor).^13–16

Chemically dimerization can be formed by three types of interactions: disulfide bond, coiled-coil interaction, and transmembrane helices interactions (Figure 1), which modulate receptor pharmacology by altering ligand binding affinity and the mode of signal transduction. In classical GPCRs, binding of an allosteric modulator at an allosteric site can change the binding affinity and efficacy of the orthosteric agonist at the same receptor, occurring at two different sites within the same receptor. Homodimerization facilitates the cooperative binding and allosteric modulation of ligands for both receptors (Figure 2). During heteromerization, evidence suggests an increase in receptor activity, such as in the angiotensin II type 1 (AT₁)-bradykinin (B2) receptor complex.¹⁷ Homodimer formation often leads to the activation of effector molecules without Gαβγ subunit dissociation, while heterodimerization may facilitate coupling to new G proteins, as observed in the δ-Opioid-μ-Opioid heterodimer coupling to Gαz.^18–20 Additionally, heterodimerization can result in the coupling of β-arrestin instead of G proteins, initiating the activation of transcription factors by modulating signals.²¹ Marino et al. (2023) utilized an advanced free-energy technique known as coarse-grained metadynamics to investigate the dimerization process. This method allowed them to simulate the binding events between receptors in a realistic timeframe. They discovered symmetric and asymmetric dimeric structures for CCR5/CCR5, CXCR4/CXCR4, and CCR5/CXCR4 receptors suggesting that these dimerizations work via positive allosteric mechanisms.²²

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IMPLICATIONS OF GPCR DIMERIZATION

Signal transduction and modulation

GPCR dimerization, particularly heterodimerization, and oligomerization has the potential to modify G protein coupling and signal transduction pathways. A notable example is adenosine A1 receptor (A1R), a GPCR that regulates neuronal excitability and pain sensitivity. In cerebellar Purkinje cells, A1R forms a trimeric complex with type 1 metabotropic glutamate receptor (mGluR1). Activation of A1R dampens neuronal responses induced by glutamate through mGluR1; mGluR1 activation attenuates A1R signaling, as evidenced by changes in intracellular cAMP levels.²³ Another receptor group, the Mas-related receptors (Mrgs), is implicated in pain physiology and serves as a therapeutic target. The heterodimeric interactions of rat MrgD and MrgE alter the MrgD signaling pathway. In human embryonic kidney 293 cell lines, MrgD induces intracellular calcium ion elevation via phosphorylation of the MAP kinases ERK1 and ERK2 in response to beta-alanine, followed by translocation to intracellular vesicles from the plasma membrane. In contrast, MrgE did not respond to beta-alanine. The co-expression of MrgD and MrgE in peripheral nociceptive neurons reveals their heterodimerization, which attenuates MrgD internalization and trafficking, underscoring the significance of dimerization in MrgD function.²⁴ Signal transduction change is also observed in the dimers and oligomers in the renin-angiotensin system (RAS) in angiotensin II receptors (AT₁R and AT₂R), Mas receptor, relaxin receptors, and adiponectin receptors which is discussed later in this review article.

Hormone resistance

Oligomerization has implications for hormone resistance disorders, such as thyroid stimulating hormone (TSH) resistance in congenital hypothyroidism.^25,26 Partial TSH resistance has been observed in cases with heterozygous mutations in the TSH receptor gene.²⁷ However, the underlying molecular mechanisms remain elusive until Calebiro et al. (2005) elucidated the dominant negative effect of mutant TSH receptors on wild-type TSH receptors. This discovery emerged from experiments in which COS-7 cells were co-transfected with wild-type and mutant TSH receptors from TSH-resistant patients. Through FRET and immunoprecipitation studies, oligomerization of these two receptor types in the endoplasmic reticulum was confirmed, shedding light on the basis of TSH resistance.²⁶

Affinity and efficacy of binding

Although pharmacological actions might be reduced in the dimerized state, dimerization can enhance affinity and signaling efficacy through synergism (e.g., μ-δ opioid receptor heterodimers, κ-δ heterodimer, and SSTR5-SSTR1 heterodimer).^18,28,29 Heterodimerization also influences receptor trafficking properties, such as surface expression (e.g., GABA_BR1a and GABA_BR2), and can lead to either increased (β2-adrenoceptor-δ opioid heterodimer) or decreased (β2-adrenoceptor-κ opioid heterodimer) agonist-mediated endocytosis,²⁸ as well as reduced ligand binding (e.g., A1-D1 heterodimer).³⁰

DRUG DISCOVERY TARGETS

GPCR dimerization is now accepted with some skepticism when a large number of experiments have shown evidence of modulation of receptor functions and the signaling process using the dimerization state- which makes a platform for drug discovery. For example, Adenosine A₁ and A_2A receptors have opposite signaling pathways that are activated by adenosine binding. These two receptors form hetero dimers in the cortical limbic and thalamic regions of the brain, respectively. At low concentration adenosine inhibits glutamate release by activating A1 receptors, whereas at high concentration facilitates the glutamate release from presynaptic neurons by activating the A_2A receptor that has opposite action to its dimer brother A₁ receptor.³¹ This dimeric state can be a tool for the dose-dependent use of A₁ receptor agonists to control glutamate release in diseases such as bipolar disorder and depression. Similarly, new compounds are being discovered as dual agonists of heterodimer complexes, for example, G_q/11 coupled D1-D2 receptor complex gets activated by the dual agonist SKF83959, which is a full agonist for D1 and a partial agonist for D2 receptors.³² This simultaneous activation is mandatory for G-protein coupling and intracellular calcium release by this oligomeric complex. Likewise, Vendrell et al. (2007) developed an ergopeptide derivative capable of acting as an agonist for the adenosine receptor and antagonist of the dopamine receptor,³³ which can be used to investigate adenosine-dopamine heterodimers. A TMD4 peptide-inhibiting dimer in the chemokine receptor CXCR4 has shown potential for targeting some inflammatory conditions.³⁴ With advances in pharmacology, biased pathways (e.g., β arrestin) can be activated using biased ligands. Gα_q, Gα_i2, Gα₁₂, and Gα₁₃ activities can be reduced by stimulating Orexin-1/Cholecystokinin A receptor heterodimers using their double ligands.³⁵ Thus it would not be an exaggeration to say that, when clinical studies are being performed on a new drug candidate, the consideration of receptor dimerization might open new targets and increase the specificity of drugs at the molecular level. In addition, state-of-the-art techniques such as co-immunoprecipitation, Western blotting, bioluminescence or fluorescence resonance energy transfer (BRET or FRET), receptor labeling, microscopy, proximity ligation assay (PLA), and protein fragment complementation assay (PCA) are used to identify homo and heterodimers and drug discovery targets.^36–42

TARGETING DIMERIZATION IN RENIN-ANGIOTENSIN SYSTEM (RAS)

AT₁ receptor homo dimerization

Angiotensin II Type I (AT₁) receptors, found in the kidney, heart, liver, lung, brain, and vascular system,⁴³ are implicated in kidney injury, blood pressure control, and fluid homeostasis, and can form dimers, initially which was thought to be protein aggregates (Milligan and Bouvier, 2005; Siemens et al., 1991; Rondeau et al., 1990). The BRET study and regulated secretion and aggregation assay (RPD)⁴⁴ showed that AT₁ receptors form constitutive homo-dimers and oligomers in COS-7 cells. This BRET signal was independent of the agonist angiotensin II agonist and the inverse agonist telmisartan. Simultaneously, the regulated secretion and aggregation technology enabled them to regulate the escape of AT₁ receptors to the cell surface from the endoplasmic reticulum (ER) by fluorescent Fm protein tagging to introduce AT₁-Fm aggregation. Treatment with the aggregation attenuator drug AP21998 revealed that surface expression of AT₁ receptors can be controlled with this drug if AT₁ is co expressed with mutated (in DRY motif) AT₁ receptor tagged to the Fm protein.⁴⁵ This study also revealed that wild-type AT₁, and mutant AT₁ receptor dimerization lose the capacity to couple Gαq but can preserve ERK activation, indicating the importance of homodimerization in AT₁ receptor activity. Similarly, AT₁ homodimerization in monocytes has been used as a diagnostic tool for atherogenesis. The intracellular factor XIIIA transglutaminase creates cross-linking with the carboxy terminal end of the AT₁ homodimer via glutamine in the presence of angiotensin II, showing desensitization and modulation of receptor signaling.⁴⁶ Inhibition of ACE and XIIIA transglutaminase showed less cross-linked AT₁ receptors in an ApoE deficient mouse atherosclerosis model, which opens a window for screening drug molecules for treating arterial diseases.^47–49 Thus, targeting AT₁ homodimers could be a therapeutic intervention for various disease conditions.

AT₂ receptor homodimerization

Angiotensin II type II (AT₂) receptors are expressed in the tissues of various organs such as the fetus, kidney, heart, lung, adrenal cortex, uterus, and vascular smooth muscle.⁴³ AT₂ receptors are involved in reducing hypertension, metabolic dysfunction, inflammation, and organ remodeling.^50,51 They reduce tubular damage by reducing the infiltration of IL-6, TNF-α, and CD11b⁺ leukocytes in acute kidney injury in renal epithelial cells.⁵² They also decrease PKC activation and superoxide formation in neuronal cells and enhance NO availability and cerebral vasorelaxation in ischemic stroke.^53,54 AT₂ receptors can form constitutive dimers with two cysteines (at the 35N terminus and 290 position in ECL3), which are crucial for dimerization.⁵⁵ Mutations in these positions can halt signaling, which might affect the protective actions of AT₂ receptors in the body.^56,57

AT₁/B2 heterodimerization

Vasoactive AT₁ and Bradykinin (B2) receptor heterodimers have been found ubiquitously in number of several cell lines, platelets, neurons, and vascular smooth muscle.^17,58 While the AT₁ receptor is related to hypertension and cardiovascular pathophysiology, the AT₁/B2 dimer showed hyper-responsiveness in the AT₁ agonist Ang II- mediated coupling in Gα_q/11 and elevated secretion of endothelin-1 from mesangial cells in a hypertensive rat model.⁵⁸ This dimer has also been found in hypertensive preeclamptic patients in a clinical settings of 30 pregnant women including 19 preeclampsia patients.⁵⁹ This AT₁/B2 dimer state correlates with Ang II hypersensitivity and a 5-fold increase in B2 protein expression, which makes this AT₁/B2 dimers a selective target for new drug candidates in hypertension and pre-eclampsia.

AT₂/B2 heterodimerization

Abadir et al. (2006) found the presence of a constitutive AT₂/B2 heterodimer in PC12W cells showing dimer entanglement in the NO production mechanism. This heterodimer also contributes to the phosphorylation of c-Jun N-terminal kinase, κBα, phosphotyrosine phosphatase, and dephosphorylation of P42/44 mitogen activated protein kinase and P38.⁶⁰ As these proteins are well known for different physiological processes, they provide a chance for the therapeutic intervention of AT₂ and B2 receptor expression in cardiovascular and renal diseases.

AT₁/AT₂ heterodimerization

AT₁ and AT₂ receptors show opposite effects in the renin-angiotensin system by antagonizing each other's signal transduction.⁶¹ The AT₁/AT₂ heterodimer is functionally relevant to the signaling pattern. Ang II forms complex with the AT₁/AT₂ heterodimer in the LLC-PK1 cell membrane and this complex is internalized through the microtubule-dependent endocytic pathway and localizes in the ER. This process also stimulates the intracellular Ca⁺² response by elevating sarcoplasmic reticulum Ca⁺²-ATPase activity.⁶² Hence, the ER can be a target organelle for RAS (Table 1).

TABLE 1 Dimers in renin-angiotensin system, and their characteristics.

Name of dimers/oligomer	Characteristics	Condition when dimer/oligomer might form	Organ/Cells/Animal model
AT1R/AT1R	AT1 receptor dimerization loses the capacity to couple Gαq but can preserve ERK activation45	Normal, Atherogenesis48	Human kidney, heart, liver, lung, brain, and vascular system43,47
AT2R/AT2R	Enhances AT2R mediated signaling and protection55	Normal condition	Human fetus, kidney, heart, lung, adrenal cortex, uterus, and vascular smooth muscle43
AT1R/B2R	Increases AngII hypersensitivity in Gαq/11 coupling58	Hypertension,58 pre-eclampsia59	Human vascular smooth muscle, platelet, neurons17,58
AT2R/B2R	Increases NO production, phosphorylation of JNKs, κBα, phosphotyrosine phosphatase, and dephosphorylation of P42/44 MAPK and P3860		PC12W cell line60
AT1R/AT2R	Stimulates the intracellular Ca+2 response by elevating sarcoplasmic reticulum Ca+2-ATPase activity62	Normal condition especially in the presence of Ang II	LLC-PK1 cell62
AT2R/MasR	Enhances receptor functions, shows functional interdependence and crosstalks12	Obesity	Obese Zucker rat kidney12
AT2R/RXFP-1	Regulates the anti-fibrotic effect of relaxin hormone after the dimer is formed63	In fibrotic conditions like CKD	Mouse model of unilateral ureteral obstruction63
AT1R/AT2R/RXFP-1	AT1R regulates the anti-fibrotic actions of AT2R or RXFP-164	Renal and cardiac fibrosis	Cardiac and renal myofibroblasts64
AT1R/Adipo R1 and AT2R/Adipo R2	Induce tubulointerstitial fibrosis65	Diabetic kidney disease, high glucose	Renal proximal tubular epithelial cells (NRK-52E)65

AT₂/Mas heterodimerization

AT₂ and Mas receptors showed functional cross-talks and physical heterodimerization in obese Zucker rat kidneys, which was confirmed by co-IP, antibody labeling, confocal microscopy, and immunoblotting.^12,66 Patel et al. (2017) showed that the AT₂R agonist drug C21 promoted natriuretic function in an obese Zucker rat model, as confirmed by increased sodium and urine flow which was lowered by concomitant administration of the AT₂R antagonist PD123319 or MasR antagonist A-779 drugs. Similar results were observed with the infusion of MasR agonist Ang-(1-7) and prior infusion of PD123319 or A-779. This heterodimerization, cross talks, and reciprocal modulation of AT2 and MasR have made them potential targets for new drug molecules such as C21 and it's possible derivatives.

AT₂/RXFP-1 heterodimerization

Relaxin hormone reduces fibrosis mediated by transforming growth factor-β1 (TGF-β1) by repealing Smad-2 phosphorylation via relaxin family peptide receptor −1 (RXFP-1). Angiotensin II also reduces the TGF- β1 activity via AT₂R. Previous studies showed anti-fibrotic action by AT₂R activation in the kidney, lung, heart, skin, and pancreas.⁶⁷ Interestingly, heterodimerization of AT₂R/RXFP-1 has been identified using both animal and in-vitro models.⁶³ The AT₂R blocker PD123319 blocked the anti-fibrotic effect of relaxin in a mice model of unilateral ureteral obstruction,⁶³ indicating the importance of AT₂R/RXFP-1 dimer in the anti-fibrotic action of relaxin in chronic kidney disease (CKD). A recent study revealed that relaxin also inhibits NLRP3 inflammasome in cardiac myofibroblast via AT₂R and ATP receptors (P2X7).⁶⁸ The existence of AT₁R/AT₂R/RXFP-1 oligomer in cardiac and renal myofibroblasts and the loss of AT₂R or RXFP-1 mediated anti-fibrotic actions by AT₁R blocker irbesartan and candesartan suggested therapeutic interventions targeting these oligomers.⁶⁴

AT₁ and AT₂/Adiponectin receptor heterodimerization

Adiponectin receptors (Adipo R1 and Adipo R2) show higher expression in diabetic kidney disease by forming heterodimers with the AngII receptors: AT₁/Adipo R1 and AT₂/Adipo R2 in renal proximal tubular epithelial cells (NRK-52E) in response to high glucose.⁶⁵ These heterodimers induced tubulointerstitial injury in kidney. Hence, targeting these heterodimers might have therapeutic potential in kidney diseases.

CONCLUDING REMARKS

GPCR dimerization or oligomerization changes signaling processes, which creates a big question: should we not reconsider receptor pharmacology in drug discovery? Although the dimerization concept has not yet gained interest in the pharmaceutical industry, there is enormous potential for finding new dimers or oligomeric targets for treating life-threatening diseases such as hypertension or atherosclerosis. Targeting biased signaling from GPCR dimers might give more information about disease conditions, cross-talks among GPCRs, and any possible side effects. Hence, GPCR dimerization studies can be worthy of consideration in drug discovery.

AUTHOR CONTRIBUTIONS

TF: conceptualization, writing, review, and editing the manuscript, editing; TH: conceptualization, review, and editing the manuscript, funding acquisition.

ACKNOWLEDGMENTS

This work is supported by National Institutes of Health R01 grants DK061578 and DK117495.

CONFLICT OF INTEREST STATEMENT

The authors declare that there is no potential financial or commercial conflict of interest with this article.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the studies mentioned in the references, and the authors can also provide any data upon request.

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