| pubmed-article:20946927 | pubmed:abstractText | Adrenomedullin 1 (AM(1)) receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) - a family B G protein-coupled receptor (GPCR) - and receptor activity-modifying protein 2 (RAMP2). Both family A and family B GPCRs possess an eighth helix (helix 8) in the proximal portion of their C-terminal tails; however, little is known about the function of helix 8 in family B GPCRs. We therefore investigated the structure-function relationship of human (h)CLR helix 8, which extends from Glu430 to Trp439, by separately transfecting nine point mutants into HEK-293 cells stably expressing hRAMP2. Glu430, Val431, Arg437 and Trp439 are all conserved among family B GPCRs. Flow cytometric analysis revealed that Arg437Ala or Trp438Ala mutation significantly reduced cell surface expression of the receptor complex, leading to a ?20% reduction in specific (125)I-AM binding but little change in their IC(50) values. Both mutants showed 6-8-fold higher EC(50) values for AM-induced cAMP production and ?50% reductions in their maximum responses. Glu430Ala mutation also reduced AM signaling by ?45%, but surface expression and (125)I-AM binding were nearly the same as with wild-type CLR. Surprisingly, Glu430Ala and Val431Ala mutations significantly enhanced AM-induced internalization of the mutant receptor complexes. Taken together, these findings suggest that within hCLR helix 8, Glu430 is crucial for Gs coupling, and Arg437 and Trp439 are involved in both cell surface expression of the hAM(1) receptor and Gs coupling. Moreover, the Glu430-Val431 sequence may participate in the negative regulation of hAM(1) receptor internalization, which is not dependent on Gs coupling. | lld:pubmed |
