Human-Xenopus chimeras of Gs alpha reveal a new region important for its activation of adenylyl cyclase.
G proteins are heterotrimeric GTPases that play a key role in signal transduction. The alpha subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gs alpha subunits are 92% identical to those found in the short form of human Gs alpha. Despite this high homology, the X. laevis Gs alpha clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc- membranes in contrast with human Gs alpha similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gs alpha. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gs alpha. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTP gamma and isoproterenol. The results obtained here clearly indicate that the G alpha sequence that extends from amino acid 70 to 140, is important for the functionality of human Gs alpha in activating adenylyl cyclase.
Antonelli
M
M
Departamento de Bioquímica, Facultad de Medicina, Universidad de Chile, Santiago.
Birnbaumer
L
L
Allende
J E
JE
Olate
J
J
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases