Identification of alpha-subunits of trimeric GTP-binding proteins in human platelets by RT-PCR
van Willigen, G.; Donath, J.; Lapetina, E.G.; Akkerman, J.W.
Biochemical and Biophysical Research Communications 214(1): 254-262
1995
ISSN/ISBN: 0006-291X PMID: 7545394 Document Number: 443194
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 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.