Abstract
BACKGROUND AND PURPOSE & para;& para;Some histamine H-4 receptor ligands act as inverse agonists at the human H-4 receptor (hH(4)R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H-4 receptor (mH(4)R) and rat H-4 receptor (rH(4)R). To study molecular determinants of constitutive activity, H-4 receptor ...
Abstract
BACKGROUND AND PURPOSE & para;& para;Some histamine H-4 receptor ligands act as inverse agonists at the human H-4 receptor (hH(4)R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H-4 receptor (mH(4)R) and rat H-4 receptor (rH(4)R). To study molecular determinants of constitutive activity, H-4 receptor reciprocal mutants were constructed: single mutants: hH(4)R-F169V, mH(4)R-V171F, hH(4)R-S179A, hH(4)R-S179M; double mutants: hH(4)R-F169V+S179A, hH(4)R-F169V+S179M and mH(4)R-V171F+M181S.& para;& para;EXPERIMENTAL APPROACH & para;& para;Site-directed mutagenesis with pVL1392 plasmids containing hH(4) or mH(4) receptors were performed. Wild-type or mutant receptors were co-expressed with G alpha(i2) and G beta(1)gamma(2) in Sf9 cells. Membranes were studied in saturation and competition binding assays ([H-3]-histamine), and in functional [S-35]-GTP gamma S assays with inverse, partial and full agonists of the hH(4) receptor.& para;& para;KEY RESULTS & para;& para;Constitutive activity decreased from the hH(4) receptor via the hH(4)R-F169V mutant to the hH(4)R-F169V+S179A and hH(4)R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH(4) receptor. Partial inverse hH(4) receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH(4) receptor agonists showed decreased maximal effects and potencies at hH(4)R-F169V and double mutants. However, the mutation of S179 in the hH(4 )receptor to M as in mH(4) receptor or A as in rH(4) receptor did not significantly reduce constitutive activity.& para;& para;CONCLUSIONS AND IMPLICATIONS & para;& para; F169 and S179 are key amino acids for the high constitutive activity of hH(4) receptors and may also be of relevance for other constitutively active GPCRs.
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