Identification of α-Subunits of Trimeric GTP-Binding Proteins in Human Platelets by RT-PCR

Abstract In a search for new α-subunits of trimeric GTP-binding proteins in human platelets, we prepared leucocyte-free platelet concentrates and analyzed total RNA for areas homologous to known α-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 α. Following subcloning in pGEM-T vector and sequencing, we identified the α-subunits G i α-2 and G s α-S of the regulating GTP-binding proteins of adenyl cyclase as well as G z α whose function is unknown, confirming earlier immunological identification. In addition, we identified G s α-L (differing from G s α-S by an insertion of 45 base pairs), G 16 α, (a member of the pertussis toxin insensitive G q -family), and two new variants of both G s α-S and G s α-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 α lead to an insertion of a Ser-residue, 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.