G protein beta 5 subunit interactions with alpha subunits and effectors.
When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.
Yoshikawa
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DM
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Hatwar
M
M
Smrcka
A V
AV
D000246:Adenosine Diphosphate Ribose / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D054799:Phospholipase C beta; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020710:RGS Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
D003902:Detergents; D004791:Enzyme Inhibitors; C118981:G-protein Beta gamma; C410342:GNG2 protein, human; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C094668:Gnb5 protein, mouse; D007527:Isoenzymes; D010750:Phosphoproteins; D020710:RGS Proteins; D011994:Recombinant Proteins; C097187:regulator of G-protein signalling 19; D000246:Adenosine Diphosphate Ribose; D006639:Histidine; D010738:Type C Phospholipases; C518033:PLCB1 protein, human; C518036:PLCB2 protein, human; C518039:PLCB3 protein, human; D054799:Phospholipase C beta; C518034:Plcb1 protein, mouse; C518037:Plcb2 protein, mouse; C518040:Plcb3 protein, mouse; D014357:Trypsin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins