Interaction between the C5a receptor and Gi in both the membrane-bound and detergent-solubilized states.
C5a elicits a variety of responses from the polymorphonuclear leukocyte all of which utilize G proteins as transducing elements. In the present study, we report the consequences of the interaction between the C5a receptor and the G proteins and describe a system which may allow identification of the transducing proteins. C5a binding to polymorphonuclear leukocyte membranes is inhibited by pertussis, but not cholera, toxin and by a variety of guanine nucleotides. In the absence of nucleotide, we observed a single class of sites with a Kd of 17 pM. The presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) did not alter this affinity but did result in a concentration-dependent decrease in the number of binding sites. Surprisingly, we did not observe the concomitant appearance of a low affinity state implying that, if such a state exists, its affinity is below our limit of detection (5 nM). The receptor and G protein retained their functional interaction following solubilization of the membrane in digitonin. In the absence of nucleotide, we observed a single class of sites with a Kd of 28 pM. Addition of GTP gamma S suppressed binding, and, as was found in membranes, this inhibition is due almost entirely to a decrease in the number of sites. Again we failed to detect the appearance of a lower affinity state. Gel filtration studies of the detergent-solubilized receptor and receptor-C5a complexes indicate that the receptor is precoupled to G protein in the absence of ligand (C5a).
Siciliano
S J
SJ
Department of Immunology Research, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Rollins
T E
TE
Springer
M S
MS
D001665:Binding Sites / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology; D002850:Chromatography, Gel; D015936:Complement C5a / Q000378:metabolism; D004072:Digitonin; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D009504:Neutrophils / Q000378:metabolism*; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D013696:Temperature; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D006150:Guanine Nucleotides; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015936:Complement C5a; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D004072:Digitonin