RGS11 interacts preferentially with R7BP over Galpha(oa)--characterization of Gbeta5-free RGS11.
Regulator of G protein signaling 11 (RGS11) is the least characterized member of the R7 family of Ggamma-like GGL domain-containing RGS proteins. All R7-RGS proteins of a variety of cell types are found in Gbeta5-containing complexes that exhibit a number of unique functional properties. However, presence of Gbeta5 reduced the affinity of R7-RGS7 for Galpha subunits, also only RGS7 bound to Muscarinic M3-Receptor, but the Gbeta5-RGS7 dimer did not, making it difficult to study differential interaction of R7-RGS proteins. Here, we report the successful purification of functionally intact, Gbeta5-free recombinant RGS11 (rRGS11), obtained by expressing N- and C-terminally truncated form of RGS11 in Escherichia coli BL 21 (DE3), that differentially interact with R7BP and Galpha(oa). rRGS11 was capable of interacting with Galpha(oa) and R7BP (RGS7 family binding protein) with equilibrium dissociation constants (K(D)) of 904 (+/- 208) nM, and 308 (+/- 97) nM, respectively. It also induced several-fold increase in the GTPase activity of Galpha(oa). The binding of rRGS11 was differential with a binding preference for R7BP over Galpha(oa) implying extended roles of R7BP. In addition, we identified a novel interaction between Galpha(oa) and R7BP with a K(D) of 592 (+/- 150) nM. The production of stable and functional rRGS11 would provide chances to discover more functions of RGS11 yet to be identified.
Saleem
Yasar
Y
Center for Neural Science, Korea Institute of Science and Technology (KIST), P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea.
Kim
Key-Sun
KS
D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D017510:Protein Folding; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; C490611:GNB5 protein, human; D044387:GTP-Binding Protein beta Subunits; D047908:Intracellular Signaling Peptides and Proteins; D020710:RGS Proteins; C400195:RGS11 protein, human; C542323:RGS7BP protein, human; D011994:Recombinant Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go