Domains of the human neutrophil N-formyl peptide receptor involved in G protein coupling. Mapping with receptor-derived peptides.
Chemotactic signaling by the human neutrophil N-formyl peptide receptor requires its association with heterotrimeric G protein. Synthetic peptides and a fusion protein derived from the intracellular regions of the receptor were used to identify sites which interact with G protein. A peptide derived from the second intracellular loop (C12R), and peptides (F15R and S22L) and a fusion protein derived from the receptor's carboxyl terminus inhibited binding of anti-Gi alpha antibody (R16,17) to Gi alpha in a competitive enzyme-linked immunoassay, and antagonized pertussis-toxin catalyzed ADP-ribosylation of Gi alpha. C12R also inhibited G protein-dependent, high affinity ligand binding to the receptor and physical coupling of receptor to G protein. In contrast, a peptide consisting of the entire third loop of the N-formyl peptide receptor was totally inactive in these assays. Collectively, these data suggest that the second intracellular loop and the carboxyl-terminal tail are important for effective N-formyl peptide receptor/G protein coupling, but that the third intracellular loop is less important in coupling, unlike previous findings with other G protein-coupled receptor systems. The chemoattractant receptor family may rely on different structural determinants to interact with GTP-binding proteins.
Schreiber
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RE
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Prossnitz
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ER
Ye
R D
RD
Cochrane
C G
CG
Bokoch
G M
GM
D000595:Amino Acid Sequence; D001665:Binding Sites; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*
D010455:Peptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019204:GTP-Binding Proteins