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National audience; Most G-protein-coupled receptors (GPCRs) form di(oligo)-meric structures that constitute signaling and trafficking units and might be essential for receptor functions. Cell responses to complement C5a receptor (C5aR) are tightly controlled by receptor desensitization and internalization. To examine the implication of dimerization in C5aR regulation, we generated an NH(2)-terminally modified C5aR mutant, unable to bind C5a, and a phosphorylation-deficient mutant. Neither an intact NH(2) terminus nor the presence of COOH-terminal phosphorylation sites appeared to be required for the formation of C5aR dimers. Upon C5a stimulation, mutant receptors did not internalize when individually expressed. C5a stimulation of cells that co-expressed wild type C5aR together with either unliganded or phosphorylation-deficient mutant resulted in co-internalization of mutant receptors with C5aR. Unliganded GPCRs can be cross-phosphorylated within a heterologous receptor dimer or by second messenger-activated kinases. C5a stimulation of (32)P-labeled cells that co-expressed the unliganded mutant with either C5aR or the phosphorylation-deficient mutant did not induce phosphorylation of the unliganded mutant. We can thus postulate that, in the case of C5aR, the stimulation and phosphorylation of one monomer is enough to lead to dimer internalization. The existence and functional implication of di(oligo)mer formation may be important for an accurate C5aR down-regulation in pathological conditions.

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.

Leukocyte recruitment to sites of inflammation and infection is dependent on the presence of a gradient of locally produced chemotactic factors. This review is focused on current knowledge about the activation and regulation of chemoattractant receptors. Emphasis is placed on the members of the N-formyl peptide receptor family, namely FPR (N-formyl peptide receptor), FPRL1 (FPR like-1) and FPRL2 (FPR like-2), and the complement fragment C5a receptors (C5aR and C5L2). Upon chemoattractant binding, the receptors transduce an activation signal through a G protein-dependent pathway, leading to biochemical responses that contribute to physiological defense against bacterial infection and tissue damage. C5aR, and the members of the FPR family that were previously thought to be restricted to phagocytes proved to have a much broader spectrum of cell expression. In addition to N-formylated peptides, numerous unrelated ligands were recently found to interact with FPR and FPRL1. Novel agonists include both pathogen- and host-derived components, and synthetic peptides. Antagonistic molecules have been identified that exhibit limited receptor specificity. How distinct ligands can both induce different biological responses and produce different modes of receptor activation and unique sets of cellular responses are discussed. Cell responses to chemoattractants are tightly regulated at the level of the receptors. This review describes in detail the regulation of receptor signalling and the multi-step process of receptor inactivation. New concepts, such as receptor oligomerization and receptor clustering, are considered. Although FPR, FPRL1 and C5aR trigger similar biological functions and undergo a rapid chemoattractant-mediated phosphorylation, they appear to be differentially regulated and experience different intracellular fates.