title: Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling.
abstract: Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. 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-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.
authors.affiliation: University Research Centre for Neuroendocrinology, University of Bristol, Bristol, BS2 8HW, United Kingdom.
authors.forename: J N
authors.initials: JN
authors.lastname: Hislop
authors.forename: H M
authors.initials: HM
authors.lastname: Everest
authors.forename: A
authors.initials: A
authors.lastname: Flynn
authors.forename: T
authors.initials: T
authors.lastname: Harding
authors.forename: J B
authors.initials: JB
authors.lastname: Uney
authors.forename: B E
authors.initials: BE
authors.lastname: Troskie
authors.forename: R P
authors.initials: RP
authors.lastname: Millar
authors.forename: C A
authors.initials: CA
authors.lastname: McArdle
mesh_terms: D000818:Animals; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011966:Receptors, LHRH / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013045:Species Specificity
chemical_list: D007295:Inositol Phosphates; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases; D034281:Dynamins