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. 1991 Feb 1;88(3):971–975. doi: 10.1073/pnas.88.3.971

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex.

T E Rollins 1, S Siciliano 1, S Kobayashi 1, D N Cianciarulo 1, V Bonilla-Argudo 1, K Collier 1, M S Springer 1
PMCID: PMC50936  PMID: 1899485

Abstract

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.

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Selected References

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