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 Gs alpha. Following subcloning in pGEM-T vector and sequencing, we identified the alpha-subunits Gi alpha-2 and Gs alpha-S of the regulating GTP-binding proteins of adenyl cyclase as well as Gz alpha whose function is unknown, confirming earlier immunological identification. In addition, we identified Gs alpha-L (differing from Gs alpha-S by an insertion of 45 base pairs), G16 alpha, (a member of the pertussis toxin insensitive Gq-family), and two new variants of both Gs alpha-S and Gs alpha-L each containing a C-A-G triplet. With G16 we have identified another candidate for pertussis-toxin insensitive signal transduction in platelets. The C-A-G containing sequences of Gs 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.
van Willigen
G
G
Department of Haematology, University Hospital Utrecht, The Netherlands.
Donath
J
J
Lapetina
E G
EG
Akkerman
J W
JW
D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D004247:DNA; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006683:HLA-DQ Antigens / Q000235:genetics; D059866:HLA-DQ beta-Chains; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism; D012313:RNA / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism
D017931:DNA Primers; D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C057615:HLA-DQbeta antigen; D012313:RNA; D004247:DNA; D000242:Cyclic AMP; D011493:Protein Kinase C; D012194:RNA-Directed DNA Polymerase; D019204:GTP-Binding Proteins