The highly conserved DRY motif located at the end of the third transmembrane of G-protein-coupled receptors has been described as a key motif for several aspects of GPCR functions. However, in the case of the vertebrate gonadotropin-releasing hormone receptor (GnRHR), the amino acid in the third position in the DRY motif is variable. In the lamprey, a most basal vertebrate, the third amino acid of the "DRY" in lamprey (lGnRHR-1) is His, while it is most often His/Gln in the type II GnRHR. To investigate the functional significance of the substitution of DRY to DRH in the GnRHR-1, second messenger signaling, ligand binding and internalization of the wild-type and mutant lGnRH receptors were characterized with site-directed mutagenesis. Treatment of the DRE(151) and DRS(151) mutant receptors with lamprey GnRH-I significantly reduced inositol phosphate compared to wild-type (DRH(151)) and DRY(151) receptors. The LogIC(50) of wild-type receptor (-9.554+/-0.049) was similar to the LogIC(50) of DRE(151), DRS(151) and DRX(151) mutants, yet these same mutants were shown to significantly reduce cell-surface expression. However, the DRY(151) mutant compared to the wild-type receptor increased cell-surface expression, suggesting that the reduction of IP production was due to the level of the cell-surface expression of the mutant receptors. The rate of internalization of DRX(151) (35.60%) was reduced compared to wild-type and other mutant receptors. These results suggest that His(151) of the lamprey GnRH receptor-1 may play a critical role in the retention of a certain level of cell-surface expression for subsequent cellular second messenger events.
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