A receptor for human anaphylatoxin C3a on HL60 cells
The anaphylatoxin, C3a, generated from C3 during complement activation, exhibits a wide range of biological activities which suggest it is a potent inflammatory and immunoregulatory mediator. These actions are thought to be mediated via specific cell surface receptors; however, only guinea pig platelets have been shown, conclusively, to express receptors recognising C3a. The objective of this project was to identify a human cell type expressing C3a receptors and to characterise that receptor. In order to characterise the human C3a receptor, relatively large quantities of human C3a had to be purified. Initially, C3a was prepared from a tryptic digest of C3, which had been isolated from human plasma. However, a heterogeneous mixture of predominantly inactive C3a-like polypeptides was obtained. Latterly, C3a was prepared directly from complement-activated serum, which provided a pure sample of intact protein. C3a produced by this second method was radio-iodinated and used to characterise the C3a receptor on the human promyelocytic cell line, HL60. Characterisation of the receptors on the differentiated cell line indicated that 125l-C3a binding was rapid and temperature dependent, apparently resulting in ligand-receptor complex internalisation and intracellular processing. This binding was shown to be saturable and specific for C3a as the inactive C3a-des-Arg was not recognised by the receptor. Together, this evidence pointed to the presence of a specific C3a receptor expressed on differentiated HL60 cells and estimated by Scatchard analysis to be present at 65000-74000 sites/cell with a Kd of 0.71-1.0 x 10-9 M. Estimates of the molecular size of the HL60 C3a receptor, by SDS/PAGE, indicated that C3a forms a 119-132 kDa complex when cross-linked to differentiated HL60 cells. Subtraction of the molecular weight of C3a indicated a size of 110-132 kDa for the receptor protein. Radio-ligand blotting experiments showed C3a to associate with two HL60 membrane proteins of molecular weights of 79-80 and 83-85 kDa. The difference in mass between cross-linked and ligand-blotted species may be due to association with a GTP-binding protein subunit in the cross-linking experiments.