title: The rat gonadotropin-releasing hormone receptor internalizes via a beta-arrestin-independent, but dynamin-dependent, pathway: addition of a carboxyl-terminal tail confers beta-arrestin dependency.
abstract: This study examined the mechanism underlying the rat GnRH receptor (GnRH-R) internalization pathway by investigating the role of added/extended C-terminal tails and the effect of beta-arrestins and dynamin. The internalization of the wild-type (WT) rat GnRH-R, stop codon mutants, GnRH-R/TRH receptor (TRH-R) chimera, rat TRH-R, and catfish GnRH-R was examined using radioligand binding assay. Overexpression of beta-arrestin in COS-7 cells expressing each of the receptor constructs substantially increased endocytosis rate constants (k(e)) of the TRH-R, catfish GnRH-R, and GnRH-R/TRH-R chimera, but not of the WT rat GnRH-R and stop codon mutants. The beta-arrestin-promoted increase in the k(e) value was diminished by cotransfecting cells with the dominant negative beta-arrestin-(319-418) mutant, whereas WT GnRH-R and stop codon mutant internalization were unaffected. Additionally, confocal microscopy showed that activated GnRH-Rs failed to induce time-dependent redistribution of either beta-arrestin-1- or beta-arrestin-2-green fluorescent protein conjugate to the plasma membrane. However, the dominant negative dynamin (DynK44A) mutant impaired internalization of all of the receptors regardless of their beta-arrestin dependency, indicating that they internalize via a clathrin-mediated pathway. We conclude that the mammalian GnRH-R uses a beta-arrestin-independent, dynamin-dependent internalization mechanism distinct from that employed by the other receptors studied.
authors.affiliation: Medical Research Council Reproductive Biology Unit, Center for Reproductive Biology, Edinburgh, United Kingdom.
authors.forename: A
authors.initials: A
authors.lastname: Heding
authors.forename: M
authors.initials: M
authors.lastname: Vrecl
authors.forename: A C
authors.initials: AC
authors.lastname: Hanyaloglu
authors.forename: R
authors.initials: R
authors.lastname: Sellar
authors.forename: P L
authors.initials: PL
authors.lastname: Taylor
authors.forename: K A
authors.initials: KA
authors.lastname: Eidne
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000502:physiology*; D002397:Catfishes; D003062:Codon; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000502:physiology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D051381:Rats; D011966:Receptors, LHRH / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605256:Arrb1 protein, rat; C000605257:Arrb2 protein, rat; D019390:Arrestins; D003062:Codon; D008164:Luminescent Proteins; D011966:Receptors, LHRH; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D013973:Thyrotropin-Releasing Hormone; D020558:GTP Phosphohydrolases; D034281:Dynamins