Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor.
The gonadotropin-releasing hormone (GnRH) receptor belongs to the superfamily of heptahelical G protein coupled receptors, most of which have a highly conserved DRYXXV/IXXPL sequence in the second intracellular (2i) loop that has been implicated in G protein coupling. The predicted 2i loop of the GnRH receptor contains serine rather than tyrosine in the DRY sequence but retains the conserved hydrophobic Leu residue, which is required for G protein coupling and internalization of muscarinic receptors. The present study examined the effects of mutating the unique Ser140 to the conserved Tyr, and the conserved Leu147 to Ala or Asp, on agonist binding, internalization, and signal transduction. 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 Leu147 mutants were indistinguishable from those of the wild-type receptor, but their internalization was reduced by about 50%. The L147A and L147D mutants also showed significant impairment of GnRH-stimulated inositol phosphate production. These findings demonstrate that substitution of Ser140 by Tyr does not affect G protein coupling but significantly increases receptor affinity and internalization rate. In contrast, replacement of a conserved aliphatic residue (Leu147) impairs both G protein coupling and agonist-induced receptor internalization.