Functional segregation of the highly conserved basic motifs within the third endoloop of the human secretin receptor

In this study, a mutagenesis-based strategy was employed to assess the role s of two highly conserved motifs (KLR and RLAR) within the third endoloop o f the human secretin receptor. Block deletion of KLRT and mutation of Lys32 3 ((KI)-I-323) significantly reduced cAMP accumulation, and these mutations did not affect ligand interaction and receptor number expressed on the cel l surface. Thus, the KLRT region at the N terminus of the third endoloop, p articularly Lys323, is important for G protein coupling. For the RLAR motif , receptors with substitutions at positions 339 and 342 from Arg to Ala (R- 339, (342)A), Glu (R-339,R- E-342), or Ile ((RI)-I-339, 342) as well as blo ck deletion of the RLAR motif were all found to be defective in both secret in-binding and cAMP production. Interestingly, a single mutation at the cor responding positions of Arg339 or Arg342 responded as the wild-type human s ecretin receptor in all functional assays, indicating that the presence of one Arg at either position within the RLAR motif is sufficient for a normal receptor function. Immunofluorescent, staining of these mutant receptors s howed that these Arg residues are responsible for surface presentation and/ or receptor stability.