Experimental evidence for lack of homodimerization of the G protein-coupled human N-formyl peptide receptor.
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.
Gripentrog
Jeannie M
JM
Department of Microbiology, Montana State University, Bozeman, MT 59717, USA.
Kantele
Katrin P
KP
Jesaitis
Algirdas J
AJ
Miettinen
Heini M
HM
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000235:genetics; D006224:Cricetinae; D019281:Dimerization; D004705:Endocytosis / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D000939:Epitopes / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000235:genetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D044042:Receptors, Formyl Peptide / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D044042:Receptors, Formyl Peptide; D001224:Aspartic Acid; D001216:Asparagine; C085706:FLAG peptide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D000409:Alanine; D002118:Calcium