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THE C-TERMINAL REGION OF THE OXYTOCIN RECEPTOR THIRD INTRACELLULAR DOMAIN CONTAIN IMPORTANT DETERMINANTS FOR G COUPLING TO PHOSPHOLIPASE C.
Yang, Ming 1, Zhang, Miao1, Sanborn, Barbara1, 1
ABSTRACT- The oxytocin receptor (OTR) third intracellular domain (3i) enhanced phosphatidylinositide (PI) turnover in a vasopressin V2 receptor (V2 R) chimera (V2 ROTR3i)(Yang et al, Endocrine Society Annual Meeting 2000, #1344). To investigate further the determinants in the OTR3i domain important for G-protein coupling to phospholipase C, receptor chimeras were generated by substituting OTR3i N(F225-A237) or C(A240-K275) terminal sequences into comparable positions in V2R using PCR. Plasmids expressing V2R and the receptor chimeras were transiently expressed in COS cells and their ability to bind 3H-Arg-vasopressin (VP) and to elicit VP- stimulated PI turnover and cAMP elevation was determined. Substitution of OTR3i N-terminal sequence resulted in a decrease in affinity for VP (Kd, V2 R: 2.2+/-0.4 nM, n=6; V2ROTR3iN: 12.4+/-0.4 nM, n=3) whereas substitution of OTR3i C-terminal sequence into V2R did not alter affinity (Kd, V2ROTR3iC: 3.6+/-0.3 nM, n=3). Under comparable conditions, V2ROTR3iC expression was slightly increased whereas V2ROTR3iN was equivalent to V2R (V2R: 210+/-4 fmole; V2ROTR3iC: 250+/-7 fmole; V2ROTR3iN: 200+/-9 fmole, n=3). Unlike V2ROTR3i, which did not significantly stimulate adenylyl cyclase in response to VP (4% of V2R response at 100 nM VP), V2ROTR3iC retained this ability, with a similar concentration-dependence and 79% of the maximal response of V2R at 100 nM VP. In contrast, the maximal response of V2ROTR3iN was only 44% of that of V2R. Strikingly, V2ROTR3iC, like V2ROTR3i, exhibited the ability to stimulate PI turnover (~4 fold over basal) when stimulated with VP, whereas V2ROTR3iN did not increase PI turnover. These data indicate that the C-terminal 35 amino acids of the OTR3i domain contains determinants that contribute to the activation of G-proteins coupling to phospholipase C and that this activity can be transferred to the V2R chimera. Supported by HD09618 and the RA Welch Foundation.
KEY WORDS: Oxytocin receptor, G protein, PI turnover, cAMP