Browsing by Author "McArdle, Craig A"

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    Asn 102 of the Gonadotropin-releasing Hormone Receptor Is a Critical Determinant of Potency for Agonists Containing C-terminal Glycinamide
    (1996) Davidson, James S; McArdle, Craig A; Davies, Peter; Elario, Ricardo; Flanagan, Colleen A; Millar, Robert P
    We demonstrate a critical role for Asn102 of the human gonadotropin-releasing hormone (GnRH) receptor in the binding of GnRH. Mutation of Asn102, located at the top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for GnRH. Eight GnRH analogs, all containing glycinamide C termini like GnRH, showed similar losses of potency between 95- and 750-fold for the [Ala102]GnRHR, compared with wild-type receptor. In contrast, four GnRH analogs that had ethylamide in place of the C-terminal glycinamide residue, showed much smaller decreases in potency between 2.4- and 11-fold. In comparisons of three agonist pairs, differing only at the C terminus, glycinamide derivatives showed an 11-20-fold greater loss of potency for the mutant receptor than their respective ethylamide derivatives. Thus Asn102 is a critical determinant of potency specifically for ligands with C-terminal glycinamide, while ligands with C-terminal ethylamide are less dependent on Asn102. These findings indicate a role for Asn102 in the docking of the glycinamide C terminus and are consistent with hydrogen bonding of the Asn102 side chain with the C-terminal amide moiety. Taken with previous data, they suggest a region of the GnRH receptor formed by the top of helices 2 and 7 as a binding pocket for the C-terminal part of the ligand.
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    Desensitization of Gonadotropin-releasing Hormone Action in αT3-1 Cells Due to Uncoupling of Inositol 1,4,5-Trisphosphate Generation and Ca 2+ Mobilization
    (1996) McArdle, Craig A; Willars, Gary B; Fowkes, Robert C; Nahorski, Stefan R; Davidson, James S; Forrest-Owen, Wyn
    Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%. Ca2+-free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P3 generation, implying that attenuation of the Ca2+ response underlies the Ins(1,4,5)P3 reduction rather than vice versa. Intracellular Ca2+ pool depletion cannot explain desensitization of the Ca2+ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (half-times 10-20 min and 4-6 h). Moreover, 1-h GnRH pre-treatment attenuated the spike Ca2+ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca2+-free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca2+ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P3 of Ca2+ from an intact intracellular pool.
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    Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors: UNCOUPLING OF DYNAMIN-DEPENDENT INTERNALIZATION FROM MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING
    (2001) Hislop, James N; Everest, Helen M; Flynn, Andrea; Harding, Tom; Uney, James B; Troskie, Brigitte E; Millar, Robert P; McArdle, Craig A
    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.