Heterologous desensitization of the chemokine receptors by the formyl peptide receptors: Calcium signaling and receptor oligomerization

Abstract

Leukocytes are exposed to a complex mix of chemotactic signals in the course of an immune response. We do not fully understand how leukocytes decode such a complex combination of signals and translate it into physiologically meaningful activity. Prior studies have outlined a process of heterologous desensitization, whereby certain chemoattractant G protein-coupled receptors (GPCRs) desensitize the function of distinct chemoattractant GPCRs. This process has been shown to involve activation of secondary messenger kinases and phosphorylation of the desensitized receptors. Furthermore, GPCRs have been shown to exist in higher order oligomeric states. How this affects the process of heterologous desensitization is currently unknown.

We address some of these issues by studying the process of formyl peptidemediated heterologous desensitization of the chemokine receptors. We have created stably-transfected HOS cells (human osteosarcoma cells), which co-express the chemokine receptor CCR5 and the FLAG-tagged formyl peptide receptor FPR. We have previously demonstrated FPR-mediated heterologous desensitization of CCR5 function in these cells. Using the newly-created HOS cells, we address the role of CCR5/FPR hetero-oligomerization in the desensitization process. We find that CCRS and FPR do not form hetero-oligomers to an appreciable degree and conclude that receptor oligomerization is not required for the process of heterologous desensitization.

Using primary human monocytes, we study the process of formyl peptide-mediated heterologous desensitization of chemokine receptor calcium signaling. We demonstrate that formyl peptides desensitize distinct chemokine receptors to a different degree. We determine that multiple chemokine receptors utilize the same calcium signaling pathway by relying on IP₃ generation through phospholipase C. We determine that rapid heterologous desensitization of the chemokine receptor CCR1 is a short-term process, which reverses within ten minutes of formyl peptide pretreatment. We also demonstrate that the magnitude of receptor desensitization does not correlate with cell surface expression levels of the desensitized receptor. Our work highlights new molecular aspects of the heterologous desensitization process and suggests that multiple signaling pathways coordinate on different time scales resulting in a two-phase process of heterologous desensitization of chemokine receptor calcium signaling.