Experimental Evidence for Lack of Homodimerization of the G Protein-Coupled Human N-Formyl Peptide Receptor

Abstract

A large number of G protein-coupled receptors have been shown to form homodimers based on a number of different techniques such as receptor coimmunoprecipitation, cross-linking, and fluorescence resonance energy transfer. In addition, functional assays of cells coexpressing a mutant receptor with a wild-type receptor have shown receptor phenotypes that can best be explained through dimerization. We asked whether the human neutrophil N-formyl peptide receptor (FPR) forms dimers in Chinese hamster ovary cells by coexpressing wild-type FPR with one of two mutants: D71A, which is uncoupled from G protein, and N297A, which has a defect in receptor phosphorylation and endocytosis. Experiments measuring chemotaxis, ligand-induced release of intracellular calcium, and p42/44 mitogen-activated protein kinase activation did not show an inhibitory effect of the coexpressed FPR D71A mutant. Coexpressed wild-type receptor was efficiently internalized, but failed to correct the endocytosis defects of the D71A and the N297A mutants. To explore the possibility that the mutations themselves prevented dimerization, we examined the coimmunoprecipitation of differentially epitope-tagged FPR. Immunoprecipitation of hemagglutinin-tagged FPR failed to coimmunoprecipitate coexpressed c-myc-tagged FPR and vice versa. Together, these data suggest that, unlike many other G protein-coupled receptors, FPR does not form homodimers.

G-Protein Coupled Receptors Dimerization: Diversity in Somatostatin Receptors Subtypes

“…In these studies, although SSTR5 was demonstrated to form both homo-and heterodimers with SSTR1 in an agonist-regulated fashion, SSTR1 remained as a monomer when expressed alone despite its activation with agonist. Consistent with several other studies, dimerization is not always a necessary mechanism in GPCR activation [119,120]. Further, subsequent studies showed that SSTR5 and SSTR1 heterodimerization was specifically induced upon activation of SSTR5 and not via activation of SSTR1 [65].…”

Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions

“…Some studies suggested a marked specificity of the interaction, e.g. k-opioid receptors (OR) dimerized with the d-OR, but not with the m-OR (Jordan & Devi 1999), others showed a promiscuous dimerization, such as the 5HT 1A serotonin receptor, which dimerized with distantly related GPCRs (Salim et al 2002), and there were negative findings as well, for example the N-formyl peptide receptor was shown not to form homodimers (Gripentrog et al 2003). Immunoprecipitation can be used to detect oligomeric GPCR complexes, as shown by a study on M2-AchR, where three differently epitope-tagged forms of the receptor (using FLAG[DYDDDDK]-, HA-, and myc-tags) were co-expressed in, and co-immunoprecipitated from Sf 9 cells (Park & Wells 2004).…”

Agonist-induced trafficking of the low-affinity formyl peptide receptor FPRL1

“…An N-terminally FLAGtagged receptor was used because an identical epitope tag had been used in a number of previous analyses of GPCR physiology, including that of the closely related formyl peptide receptor FPR, and the tag was shown not to interfere with internalization and intracellular routing [see, for example, refs. 24,25]. In the control experiments, internalization of transferrin receptors was monitored by employing fluorescently labeled transferrin.…”