Characterization of the isolated granulocyte C5a receptor

The authors previously demonstrated that the C5a receptor may be covalently labeled with a photoreactive C5a analog termed AMEA-SC5a. The resulting C5a-receptor complex exhibits a molecular weight of 52,000 Daltons on SDS-PAGE. More recently, they have been able to characterize the C5a receptor itself, rather than the C5a-receptor complex. This was achieved by specifically radioiodinating the granulocyte C5a receptor with a unique C5a photoaffinity probe (ASAMEA-SC5a) that contained on 125I-labeled p-azido-salicylate moiety. After this probe was bound to the membrane receptor and the resulting complex was photolyzed, the C5a polypeptide was released by reductive cleavage with DTT. The resulting radiolabeled receptor was then separated from free C5a and analyzed by SDS-PAGE under reducing conditions. The C5a receptor was identified as a single molecular species with an apparent molecular weight of 46,000 to 47,000 Daltons. Furthermore, addition of disuccinimidyl suberate (DSS) to preformed C5a receptor complexes resulted in relatively selective crosslinking of the C5a receptor to a second protein with an apparent molecular weight of ca. 40,000 Daltons. These findings suggest that the C5a receptor to a second protein with an apparent molecular weight of ca. 40,000 Daltons. These findings suggest that C5a receptor may consist of two functionally different subunits or it is in close proximity to other unique membrane constituents