Molecular cloning and sequence determination of four different cDNA species coding for α-subunits of G proteins from Xenopus laevis oocytes

AbstractA cDNA library preprared from Xenopus laevis oocytes in λgt10 was screened with a mixture of three oligonucleotide probes designed to detect sequences found in different mammalian genes coding for a-subunits of G-proteins. In addition to a clone coding for a Gαo-type subunit previously reported [(1989) FEBS Lett. 244, 188-192] four additional clones have been found coding for different Gα protein subunits. By comparison with mammalian α-subunits, these oocyte cDNAs correspond to two closely related Gas-la, to a Gαi-1 and to a Gαi-3 species. The derived amino acid sequences showed that both Gαs species contain 379 residues, corresponding to the short species without the serine residue and with a calculated Mr of 42720. The Gαi-1 gene encodes a 354 amino acid protein with an Mr, of 39000 and the Gαi-3 encodes an incomplete open reading frame of 345 residues, lacking the first 9 amino acid residues at the NH2, terminus. All these Gα-subunits showed high identity with their respective mammalian counterparts (75–80%), indicating a great degree of conservation through the evolution and the important cellular regulatory function that they play

Glucagon-stimulable Adenylyl Cyclase in Rat Liver Effects of Chronic Uremia and Intermittent Glucagon Administration

Astract. The effects of chronic uremia and glucagon administration on glucagon-stimulable adenylyl cyclase in rat liver were assessed by determinations of adenylyl cyclase activities, specific iodoglucagon binding, and the activity of the stimulatory regulatory component of adenylyl cyclase. Glucagon-stimulated adenylyl cyclase was reduced in uremia to 75-80 % of control levels (P < 0.05), in the presence or absence of saturating levels of guanosine triphosphate (GTP) and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. Although these changes were accompanied by a concomitant 20 % reduction in sodium fluoride-stimulated activity, basal, GTP-, GMP-P(NH)P-, and manganese-dependent adenylyl cyclase activities were unchanged. Using [125I-Tyr'0]monoiodoglucagon as a receptor probe, the number of high affinity glucagon-binding sites was reduced 28 % (P < 0.0 1) in uremic as compared with control liver membranes. However, the affinity of these binding sites was unaltered. The S49 cyC-reconstituting activity with respect to both GMP-P(NH)P- and isoproterenol plus GTP-stimulable adenylyl cyclase was unaltered in membranes from uremic as compared with control rats. Intermittent glucagon (80-100,g) injections administered at 8-h intervals to normal rats reproduced all ofthe above described effects ofchronic experimental uremia on the adenylyl cyclase system. It is concluded that changes in the hormone-stimulable ad-Address all reprint requests to Dr. Garber