IDENTIFICATION OF ALPHA-SUBUNITS OF TRIMERIC GTP-BINDING PROTEINS IN HUMAN PLATELETS BY RT-PCR

In a search for new alpha-subunits of trimeric GTP-binding proteins in human platelets, we prepared leucocyte-free platelet concentrates and analyzed total RNA for areas homologous to known alpha-subunits. RT-PCR based on two degenerate primers revealed the expected band of 495 base pairs and an additional band of 540 base pairs reflecting the alternative splice product of G(s) alpha. Following subcloning in pGEM-T vector and sequencing, we identified the alpha-subunits G(i) alpha-2 and G(s) alpha-S of the regulating GTP-binding proteins of adenyl cyclase as well as G(z) alpha whose function is unknown, confirming earlier immunological identification. In addition, we identified G(s) alpha-L (differing from G(s) alpha-S by an insertion of 45 base pairs), G(16)alpha, (a member of the pertussis toxin insensitive G(q)-family), and two new variants of both G(s) alpha-S and G(s) alpha-L each containing a C-A-G triplet. with G(16) we have identified another candidate for pertussis-toxin insensitive signal transduction in platelets. The C-A-G containing sequences of G(s) alpha lead to an insertion of a serresidue, which results in the consensus sequence of a phosphorylation site for protein kinase c (Ser-X-Lys), making these variants candidates for protein kinase C-sensitive cyclic AMP formation. (C) 1995 Academic Press, Inc.