Characterization of mammalian Gs-alpha proteins expressed in yeast.
The guanine nucleotide regulatory protein, GS, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat GS were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP1 promoter and transformed into Saccharomyces cerevisiae. In the presence of 100 microM CuSO4, the transformed yeast expressed GS-alpha mRNAs and proteins. In reconstitution experiments, rat GS-alpha(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous GS-alpha. GS-alpha (S) demonstrated twice the activity of GS-alpha(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of GS-alpha (S) expressed in yeast with GS purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified GS. Addition of bovine brain beta gamma subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for GS-alpha(S and L) obtained from yeast. In contrast, transducin beta gamma only enhanced agonist-stimulated adenylyl cyclase activity for GS-alpha (S and L) following reconstitution. These results demonstrate that the expression of functional mammalian GS-alpha subunits in yeast may be useful for their biochemical characterization.
Stadel
J M
JM
Department of Molecular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Ecker
D J
DJ
Powers
D A
DA
Marsh
J
J
Hoyle
K
K
Gross
M
M
Minnich
M D
MD
Butt
T R
TR
Crooke
S T
ST
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003300:Copper / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D015293:Transducin / Q000494:pharmacology
D008565:Membrane Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003300:Copper; D008668:Metallothionein; D019204:GTP-Binding Proteins; D015293:Transducin; D000262:Adenylyl Cyclases