Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation.
RGS proteins constitute a large family that modulates heterotrimeric G-protein signaling. We previously showed that RGS8 suppressed Gq signaling in a receptor type-specific manner. To elucidate molecular mechanisms underlying receptor-specific attenuation by RGS8, we examined whether RGS8 can interact with certain G-protein-coupled receptors. By pull-down assay, we showed that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors (mAChRs). The binding of RGS8S, a splice variant with a different N-terminus, was weaker. RGS8 could bind specifically to the C-terminal part of M1i3 (containing amino acids of 304-353 of i3 of human M1-mAChR), but RGS8S could not. Moreover, deletion of the N-terminal 9 amino acids and substitution of both Arg-8 and Arg-9 of RGS8 with Ala resulted in reduced binding to M1i3. BRET experiments revealed that RGS8 actually interacts with M1-mAChR, but RGS8S does not interact in the living cells. The RGS8 mutant, which had less binding ability to M1i3, showed a reduced inhibitory function of Gq signaling through M1-mAChR. These results demonstrated that RGS8 can directly interact with M1-mAChR via its N-terminus and the i3 loop of the receptor, and this binding must play an essential role in receptor-specific suppression by RGS8.
Itoh
Masayuki
M
Department of Bio-Science, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan.
Nagatomo
Katsuhiro
K
Kubo
Yoshihiro
Y
Saitoh
Osamu
O
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D011485:Protein Binding / Q000502:physiology; D020710:RGS Proteins / Q000235:genetics*; D043643:Receptor, Muscarinic M1 / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014981:Xenopus
D020710:RGS Proteins; C116924:RGS8 protein, human; D043643:Receptor, Muscarinic M1; D049452:Green Fluorescent Proteins; D008156:Luciferases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11