Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex.
We have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a Kd of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the alpha and beta subunits of Gi, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm our earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.
Rollins
T E
TE
Department of Immunology Research, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Siciliano
S
S
Kobayashi
S
S
Cianciarulo
D N
DN
Bonilla-Argudo
V
V
Collier
K
K
Springer
M S
MS
D002462:Cell Membrane / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D015936:Complement C5a / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology*; D037342:Pertussis Toxin; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D010566:Virulence Factors, Bordetella; D009243:NAD; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015936:Complement C5a; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins