Previous studies have indicated that the carboxy-terminus of yl is important in the interaction of Gt with light-activated rhodopsin. Membranes from CHO cells expressing muscarinic receptor isotype 2 (m2) were purified and depleted of endogenous G-proteins. GTP-y-35S incorporation stimulated by agonist-activated m2 (membranes reconstituted with G,2 -recombinant o,2 and brain βy) was inhibited by a geranylgeranylated (GG)-peptide with the sequence of the carboxyterminus of ys, whereas a GG-peptide with scrambled YI sequence was inactive. In displacement assays of [3H]-NMS, the binding affinity of m2 for the agonist carbachol was reduced ten-fold in the presence of GGwild type y, peptide (ICM of 500 nM), whereas the GG-wild type Yz peptide and GG-scrambted y5 peptide were weak or inactive respectively. The binding affinity for the antagonist QNB was reduced in the presence of GG-wiU type YJ peptide, yet for scopolamine and atropine the affinities were unchanged. These results indicate that the interaction with the GG-wild type YJ peptide gives rise to a novel receptor state with distinct ligand-binding properties. We think that the interaction with the carboxy-termina] domain of Ys stabilizes a conformational change on the m2 receptor that is important for coupling of G proteins to receptors.
|State||Published - Dec 1 1998|