Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex (complement component C5/affinity chromatography/reversible modification/guanine nucleotide-binding protein)

We have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purifi'cation 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 witlh a Kd of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purifi'ed receptor contained a guanine nucleotide-binding pro- tein (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 a and j3 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 mole- cule. 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.