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The G protein-coupled receptor kinase family comprises six members (GRK1 to GRK6) that phosphorylate and desensitize a number of agonist-occupied G protein-coupled receptors. Overexpression of the dominant negative mutant GRK2-K220R is often accompanied by an inhibition of the agonist-mediated phosphorylation of G protein-coupled receptors. In the case of the C5a receptor (C5aR), the overexpression of wild-type GRK2 or GRK6 as well as of catalytically inactive forms of these kinases (GRK2-K220R and GRK6-K215R) failed to increase or to inhibit the agonist-mediated phosphorylation of C5aR, respectively. Replacement of Lys215 by an arginine residue in GRK6 yielded a protein with a relative molecular mass of 63 kDa, whereas wild-type GRK6 had a relative molecular mass of 66 kDa on polyacrylamide gel. The mutations S484D and T485D in the catalytically inactive mutant GRK6-K215R resulted in a protein (GRK6-RDD) with the same electrophoretic mobility as wild-type GRK6. Furthermore, in the absence of phosphatase inhibitors, GRK6 was rapidly converted into the 63 kDa species, whereas GRK6-RDD was not. Overepression of GRK6-RDD failed to alter the agonist-mediated phosphorylation of C5aR. Taken together, the results suggest that C5aR is not a substrate for either GRK2 or GRK6 and that GRK6 is very likely autophosphorylated on Ser484 and Thr485 in vivo.

Treatment of human prothrombin by α chymotrypsin results in the production of a single homogeneous product with an apparent M.W. of 69 000. This product fails to adsorb on barium citrate. NH2-terminal sequence analysis shows that a single cleavage has occurred after tyrosine 44. The proteolytically modified prothrombin which can therefore be referred to as prothrombin (des 1–44), lacks the whole vitamin K dependent part of the molecule. The region of the peptide chain around tyrosine 44 must be particularly exposed to proteolytic attack and serve as a junction between the vitamin K dependent domain and the other structural domains of prothrombin. In the presence of factor Xa alone, prothrombin (des 1–44) is indistinguishable from normal prothrombin when activation is monitored by the appearance of amidolytic activity on S2238. However, activation of prothrombin (des 1–44) is no longer enhanced by the presence of Ca++ and phospholipid in the activation mixture.