Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors - Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling

Desensitization and internalization of G-protein-coupled receptors can refl ect receptor phosphorylation-dependent binding of beta -arrestin, which pre vents G-protein activation and targets receptors for internalization via cl athrin-coated vesicles. These can be pinched off by a dynamin collar, and p roteins controlling receptor internalization can also mediate mitogen-activ ated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimul ates internalization of its receptors via clathrin-coated vesicles. Mammali an GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails a nd do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Usi ng recombinant adenovirus expressing human and Xenopus GnRH-Rs we have expl ored the relationship between receptor internalization and mitogen-activate d protein kinase signaling in HeLa cells with regulated tetracycline-contro lled expression of wild-type or a dominant negative mutant (K44A) of dynami n. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH- R internalization but dramatically reduced internalization of Xenopus GnRH- R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediat ed internalization (sucrose) abolished internalization of all three recepto rs. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both recept ors, this was inhibited by K44A dynamin. The same was true for EGF- and pro tein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was als o inhibited by a protein kinase C inhibitor but not affected by an EGF rece ptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-d esensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated inte rnalization by functionally distinct mechanisms, b) GnRII-R signaling to ER K 2 is dynamin-dependent and c) this does not reflect a dependence on dynam in-dependent GnRH-R internalization.