Critical implication of transmembrane Phe310, possibly in conjunction with Trp279, in the rat gonadotropin-releasing hormone receptor activation

The GnRH-R belongs to the superfamily of heptahelical GPCRs. A three-dimensional model of GnRH binding to its receptor predicted that Trp3 was the most deeply buried residue, potentially allowing it to interact with both Trp279, a highly conserved residue in the TMH 6 of GPCRs, and Phe310, present essentially in TMH 7 of GnRH-Rs. Replacement of Phe310 with Leu, the most common positional residue in GPCRs, induced a slightly decreased Bmax (1.6-fold) and affinity (3.8-fold); in addition, IP production was completely abolished. Similarly, replacement of Trp279 with Ser depressed the Bmax by 5.2-fold, the affinity by 2.3-fold, and totally abrogated IP production. The effect of the double mutation was not additive on binding, since the Bmax was reduced to the level of the Phe310Leu mutant, although the Kd was restored to a value not significantly different from that of the wild-type. The double mutant was also unable to induce IP production. Unexpectedly, no influence of any single or double substitution was noted on receptor internalization. These data provide evidence for the crucial role of Phe310, possibly in conjunction with Trp279, on GnRH transduction and suggest that the conformation for phospholipase C activation may not be required for GnRH-R internalization.