G protein beta gamma subunits. Simplified purification and properties of novel isoforms.
The beta and gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) form tightly associated complexes. To examine functional differences among the large number of possible combinations of unique beta and gamma subunits, we have synthesized and characterized beta gamma complexes containing gamma 5 and gamma 7, two widely distributed gamma subunits. When either gamma 5 or gamma 7 is expressed concurrently with beta 1 or beta 2 subunits in a baculovirus/Sf9 cell system, all four subunit complexes support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 (where "r" indicates recombinant), indicating formation of functional complexes. Each of the complexes was purified by subunit exchange chromatography, using the G203A mutant of rGi alpha 1 as the immobilized ligand. The purified preparations were compared with other recombinant beta gamma subunits, including beta 1 gamma 1 and beta 1 gamma 2, for their ability to modulate type I and II adenylyl cyclase activities; stimulate phosphoinositide-specific phospholipase C beta; support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 and Go alpha; and inhibit steady-state GTP hydrolysis catalyzed by Gs alpha, Go alpha, and myristoylated rGi alpha 2. The results emphasize the unique properties of beta 1 gamma 1. The properties of the complexes containing gamma 5 or gamma 7 were similar to each other and to those of beta 1 gamma 2.
Ueda
N
N
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Iñiguez-Lluhi
J A
JA
Lee
E
E
Smrcka
A V
AV
Robishaw
J D
JD
Gilman
A G
AG
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D002147:Calmodulin; D017931:DNA Primers; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium