title: Characterization of mammalian Gs-alpha proteins expressed in yeast.
abstract: 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.
authors.affiliation: Department of Molecular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
authors.forename: J M
authors.initials: JM
authors.lastname: Stadel
authors.forename: D J
authors.initials: DJ
authors.lastname: Ecker
authors.forename: D A
authors.initials: DA
authors.lastname: Powers
authors.forename: J
authors.initials: J
authors.lastname: Marsh
authors.forename: K
authors.initials: K
authors.lastname: Hoyle
authors.forename: M
authors.initials: M
authors.lastname: Gross
authors.forename: M D
authors.initials: MD
authors.lastname: Minnich
authors.forename: T R
authors.initials: TR
authors.lastname: Butt
authors.forename: S T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: 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
chemical_list: 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