A role of Histidine151 in the lamprey gonadotropin-releasing hormone receptor-1 (lGnRHR-1): Functional insight of diverse amino acid residues in the position of Tyr of the DRY motif in GnRHR from an ancestral type II receptor

Abstract

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.

Figure 1

Sequence alignment of the proximal region of TM3 and DRY motif in GnRHR from representative vertebrate species. The alignment was arranged with the entire amino acid sequences of the receptors based on Clustal W. The accession numbers for species are followed : Lamprey (AAQ04564), Catfish1 (O42329), Catfish2 (AAM95605), Medaka1 (NP_001098352), Medaka2 (NP_001098392), Medaka3 (NP_001098393), GoldfishA (AAD20001), GoldfishB (AAD20002), Pufferfish1-1 (BAE45695),Pufferfish1-2 (BAE45697), Pufferfish1-3 (BAE45699), Pufferfish2-1 (BAE45701), Pufferfish2-2 (BAE45704), Bullfrog1 (AAG42575), Bullfrog2 (AAG42949), Bullfrog3 (AAG42574), Chicken1(NP_989984), Chicken2 (NP_001012627), RhesusMonkey2 (NP_001028014), Cow (NP_803480), Sheep (NP_001009397), Pig (NP_999438), Dog (NP_001003121), Horse (NP_001075305), Rat (NP_112300), Mouse (NP_034453), Human (NP_000397).

Figure 2

A schematic diagram of the location of the point mutaions in lGnRHR-1. The rectangle shows the DRH motif and the point substitutions and deletion in lGnRHR-1 corresponding to DRY motif in GPCR. In lGnRHR-1, Tyr of DRY is substituted with His (wild-type), same as most of type II receptors, while Tyr is substituted with Ser in type I receptor. His¹⁵¹ in lGnRHR-1 was substituted with Tyr (DRY¹⁵¹), Glu (DRE¹⁵¹), or Ser (DRS¹⁵¹) or deleted (DRX¹⁵¹).

Figure 3

IP response of wild-type and mutant lGnRHRs. DRH¹⁵¹ (wild-type), DRY¹⁵¹, DRE¹⁵¹ and DRS¹⁵¹ showed different dose-response curves responding to lGnRH-III. The values of IP productions were shown as dpm. The data are mean ± SEM of duplicate in a representative experiment. LogEC50 of DRE¹⁵¹ and DRS¹⁵¹ were higher than DRH¹⁵¹ (wild-type) while the maximal IP productions were lower than DRH¹⁵¹ (wild-type). LogEC50 of DRY¹⁵¹ was lower than DRH¹⁵¹ (wild-type) while the maximal IP production was similar to DRH¹⁵¹ (wild-type).

Figure 4

Ligand binding of wild-type and mutant lGnRHRs. Competitive binding assays of ¹²⁵I-lGnRHR-1 were performed with decreasing concentrations of lGnRH III. The values of radioligand binding in the graph were shown as the percentages of maximum ¹²⁵I-lGnRH binding (mean ± SEM in triplicate). The data represents one of three independent experiments with essentially same results. Log IC50 of wild-type and mutant receptors were similar but total bindings of ¹²⁵I-lGnRH were varied

Figure 5

Receptor expression of wild-type and mutant lGnRHRs. The data are shown as the percentages of wild-type binding to ¹²⁵I-lGnRH (1 nM) and each bar represents mean ± SEM of three independent experiments in triplicate. One-way ANOVA was performed with Bonferroni post hoc test (a, P < 0.001 vs WT ; b, P < 0.001 vs DRY )

Figure 6

Internalization of wild-type and mutant lGnRHRs. Percent internalization was expressed as the ratio of internalized radioligand to the total cell-surface radioligand at each time point. Data are shown as mean ± SEM in triplicate, representing at least two independent experiments.