EFFECTS OF 2ND INTRACELLULAR LOOP MUTATIONS ON SIGNAL-TRANSDUCTION AND INTERNALIZATION OF THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR

The gonadotropin-releasing hormone (GnRH) receptor belongs to the supe rfamily of heptahelical G protein coupled receptors, most of which hav e a highly conserved DRYXXV/IXXPL sequence in the second intracellular (2i) loop that has been implicated in G protein coupling. The predict ed 2i loop of the GnRH receptor contains serine rather than tyrosine i n the DRY sequence but retains the conserved hydrophobic Leu residue, which is required for G protein coupling and internalization of muscar inic receptors. The present study examined the effects of mutating the unique Ser(140) to the conserved Tyr, and the conserved Leu(147) to A la or Asp, on agonist binding, internalization, and signal transductio n. The S140Y mutant showed a 100% increase in agonist binding affinity , and its internalization was increased by 60% above that of the wild- type receptor. The binding characteristics of the Leu(147) mutants wer e indistinguishable from those of the wild-type receptor, but their in ternalization was reduced by about 50%. The L147A and L147D mutants al so showed significant impairment of GnRH-stimulated inositol phosphate production, These findings demonstrate that substitution of Ser(140) by Tyr does not affect G protein coupling but significantly increases receptor affinity and internalization rate. In contrast, replacement o f a conserved aliphatic residue (Leu(147)) impairs both G protein coup ling and agonist-induced receptor internalization.