Abstract
The C-terminal tail of the long splice variant of the rat thyrotropin-releasing hormone (TRH) receptor-1 (TRHR-1L) comprises around 93 amino acids. A series of C-terminal truncations was constructed and expressed transiently in HEK-293 cells. The extent of steady-state internalization of these in response to [(3)H]TRH was dependent upon the degree of truncation. Little effect was produced by deletion of the C-terminal to 50 amino acids, although there was a substantial decrease in the extent of internalization by deletion to 45-46 amino acids. The rate of internalization of TRHR-1L in response to ligand was substantially decreased by the acid-wash procedures often used in the analysis of cellular distribution of receptors with peptide ligands, and thus an alternative procedure using a Mes-containing buffer was employed in the present study. Apart from a truncation anticipated to eliminate post-translational acylation of the re-ceptor, which altered both the association and dissociation rates of [(3)H]TRH, the kinetics of ligand binding were unaffected by C-terminal truncation. Equally, the rate of recycling to the plasma membrane of internalized receptors was unaffected by C-terminal truncation. Although the extent of internalization of the full-length receptor was impaired by pre-exposure of cells to TRH, this was not true of C-terminal truncation mutants, which displayed limited steady-state internalization ratios. A mutant with a substantial C-terminal deletion also displayed decreased functional desensitization compared with the full-length receptor.
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- Anderson L., Alexander C. L., Faccenda E., Eidne K. A. Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells. Biochem J. 1995 Oct 15;311(Pt 2):385–392. doi: 10.1042/bj3110385. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aragay A. M., Katz A., Simon M. I. The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells. J Biol Chem. 1992 Dec 15;267(35):24983–24988. [PubMed] [Google Scholar]
- Ashworth R., Yu R., Nelson E. J., Dermer S., Gershengorn M. C., Hinkle P. M. Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):512–516. doi: 10.1073/pnas.92.2.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Awaji T., Hirasawa A., Kataoka M., Shinoura H., Nakayama Y., Sugawara T., Izumi S., Tsujimoto G. Real-time optical monitoring of ligand-mediated internalization of alpha1b-adrenoceptor with green fluorescent protein. Mol Endocrinol. 1998 Aug;12(8):1099–1111. doi: 10.1210/mend.12.8.0149. [DOI] [PubMed] [Google Scholar]
- Barak L. S., Ferguson S. S., Zhang J., Martenson C., Meyer T., Caron M. G. Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol. 1997 Feb;51(2):177–184. doi: 10.1124/mol.51.2.177. [DOI] [PubMed] [Google Scholar]
- Böhm S. K., Grady E. F., Bunnett N. W. Regulatory mechanisms that modulate signalling by G-protein-coupled receptors. Biochem J. 1997 Feb 15;322(Pt 1):1–18. doi: 10.1042/bj3220001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cao J., O'Donnell D., Vu H., Payza K., Pou C., Godbout C., Jakob A., Pelletier M., Lembo P., Ahmad S. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. J Biol Chem. 1998 Nov 27;273(48):32281–32287. doi: 10.1074/jbc.273.48.32281. [DOI] [PubMed] [Google Scholar]
- Clague M. J. Molecular aspects of the endocytic pathway. Biochem J. 1998 Dec 1;336(Pt 2):271–282. doi: 10.1042/bj3360271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeBlasi A., O'Reilly K., Motulsky H. J. Calculating receptor number from binding experiments using same compound as radioligand and competitor. Trends Pharmacol Sci. 1989 Jun;10(6):227–229. doi: 10.1016/0165-6147(89)90266-6. [DOI] [PubMed] [Google Scholar]
- Drmota T., Gould G. W., Milligan G. Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the thyrotropin-releasing hormone receptor. J Biol Chem. 1998 Sep 11;273(37):24000–24008. doi: 10.1074/jbc.273.37.24000. [DOI] [PubMed] [Google Scholar]
- Faussner A., Proud D., Towns M., Bathon J. M. Influence of the cytosolic carboxyl termini of human B1 and B2 kinin receptors on receptor sequestration, ligand internalization, and signal transduction. J Biol Chem. 1998 Jan 30;273(5):2617–2623. doi: 10.1074/jbc.273.5.2617. [DOI] [PubMed] [Google Scholar]
- Gershengorn M. C., Heinflink M., Nussenzveig D. R., Hinkle P. M., Falck-Pedersen E. Thyrotropin-releasing hormone (TRH) receptor number determines the size of the TRH-responsive phosphoinositide pool. Demonstration using controlled expression of TRH receptors by adenovirus mediated gene transfer. J Biol Chem. 1994 Mar 4;269(9):6779–6783. [PubMed] [Google Scholar]
- Gershengorn M. C., Osman R. Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiol Rev. 1996 Jan;76(1):175–191. doi: 10.1152/physrev.1996.76.1.175. [DOI] [PubMed] [Google Scholar]
- Heding A., Vrecl M., Bogerd J., McGregor A., Sellar R., Taylor P. L., Eidne K. A. Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem. 1998 May 8;273(19):11472–11477. doi: 10.1074/jbc.273.19.11472. [DOI] [PubMed] [Google Scholar]
- Honda N., Machida K., Mamiya T., Takahashi T., Takishima T., Hasegawa N., Kamano T., Hashimoto M., Ohno K., Hosoba M. The optimum Butterworth-Wiener filter for I-123 IMP brain SPECT. Radiat Med. 1989 May-Jun;7(3):124–128. [PubMed] [Google Scholar]
- Hsieh K. P., Martin T. F. Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. Mol Endocrinol. 1992 Oct;6(10):1673–1681. doi: 10.1210/mend.6.10.1333052. [DOI] [PubMed] [Google Scholar]
- Itadani H., Nakamura T., Itoh J., Iwaasa H., Kanatani A., Borkowski J., Ihara M., Ohta M. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochem Biophys Res Commun. 1998 Sep 8;250(1):68–71. doi: 10.1006/bbrc.1998.9268. [DOI] [PubMed] [Google Scholar]
- Kallal L., Gagnon A. W., Penn R. B., Benovic J. L. Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor. J Biol Chem. 1998 Jan 2;273(1):322–328. doi: 10.1074/jbc.273.1.322. [DOI] [PubMed] [Google Scholar]
- Kim G. D., Carr I. C., Anderson L. A., Zabavnik J., Eidne K. A., Milligan G. The long isoform of the rat thyrotropin-releasing hormone receptor down-regulates Gq proteins. J Biol Chem. 1994 Aug 5;269(31):19933–19940. [PubMed] [Google Scholar]
- Koenig J. A., Edwardson J. M. Endocytosis and recycling of G protein-coupled receptors. Trends Pharmacol Sci. 1997 Aug;18(8):276–287. doi: 10.1016/s0165-6147(97)01091-2. [DOI] [PubMed] [Google Scholar]
- Koenig J. A., Kaur R., Dodgeon I., Edwardson J. M., Humphrey P. P. Fates of endocytosed somatostatin sst2 receptors and associated agonists. Biochem J. 1998 Dec 1;336(Pt 2):291–298. doi: 10.1042/bj3360291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee T. W., Anderson L. A., Eidne K. A., Milligan G. Comparison of the signalling properties of the long and short isoforms of the rat thyrotropin-releasing-hormone receptor following expression in rat 1 fibroblasts. Biochem J. 1995 Aug 15;310(Pt 1):291–298. doi: 10.1042/bj3100291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lefkowitz R. J. G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J Biol Chem. 1998 Jul 24;273(30):18677–18680. doi: 10.1074/jbc.273.30.18677. [DOI] [PubMed] [Google Scholar]
- McGraw T. E., Maxfield F. R. Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain. Cell Regul. 1990 Mar;1(4):369–377. doi: 10.1091/mbc.1.4.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nussenzveig D. R., Heinflink M., Gershengorn M. C. Agonist-stimulated internalization of the thyrotropin-releasing hormone receptor is dependent on two domains in the receptor carboxyl terminus. J Biol Chem. 1993 Feb 5;268(4):2389–2392. [PubMed] [Google Scholar]
- Petrou C., Chen L., Tashjian A. H., Jr A receptor-G protein coupling-independent step in the internalization of the thyrotropin-releasing hormone receptor. J Biol Chem. 1997 Jan 24;272(4):2326–2333. doi: 10.1074/jbc.272.4.2326. [DOI] [PubMed] [Google Scholar]
- Pippig S., Andexinger S., Lohse M. J. Sequestration and recycling of beta 2-adrenergic receptors permit receptor resensitization. Mol Pharmacol. 1995 Apr;47(4):666–676. [PubMed] [Google Scholar]
- Sellar R. E., Taylor P. L., Lamb R. F., Zabavnik J., Anderson L., Eidne K. A. Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland. J Mol Endocrinol. 1993 Apr;10(2):199–206. doi: 10.1677/jme.0.0100199. [DOI] [PubMed] [Google Scholar]
- Svoboda P., Kim G. D., Grassie M. A., Eidne K. A., Milligan G. Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11alpha: analysis of agonist regulation of coexpressed G11alpha species variants. Mol Pharmacol. 1996 Apr;49(4):646–655. [PubMed] [Google Scholar]
- Tarasova N. I., Stauber R. H., Choi J. K., Hudson E. A., Czerwinski G., Miller J. L., Pavlakis G. N., Michejda C. J., Wank S. A. Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A. J Biol Chem. 1997 Jun 6;272(23):14817–14824. doi: 10.1074/jbc.272.23.14817. [DOI] [PubMed] [Google Scholar]
- Trapaidze N., Keith D. E., Cvejic S., Evans C. J., Devi L. A. Sequestration of the delta opioid receptor. Role of the C terminus in agonist-mediated internalization. J Biol Chem. 1996 Nov 15;271(46):29279–29285. doi: 10.1074/jbc.271.46.29279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vrecl M., Anderson L., Hanyaloglu A., McGregor A. M., Groarke A. D., Milligan G., Taylor P. L., Eidne K. A. Agonist-induced endocytosis and recycling of the gonadotropin-releasing hormone receptor: effect of beta-arrestin on internalization kinetics. Mol Endocrinol. 1998 Dec;12(12):1818–1829. doi: 10.1210/mend.12.12.0207. [DOI] [PubMed] [Google Scholar]
- Yu R., Hinkle P. M. Desensitization of thyrotropin-releasing hormone receptor-mediated responses involves multiple steps. J Biol Chem. 1997 Nov 7;272(45):28301–28307. doi: 10.1074/jbc.272.45.28301. [DOI] [PubMed] [Google Scholar]
- Yu R., Hinkle P. M. Signal transduction, desensitization, and recovery of responses to thyrotropin-releasing hormone after inhibition of receptor internalization. Mol Endocrinol. 1998 May;12(5):737–749. doi: 10.1210/mend.12.5.0110. [DOI] [PubMed] [Google Scholar]
- de la Peña P., Delgado L. M., del Camino D., Barros F. Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells. Biochem J. 1992 Jun 15;284(Pt 3):891–899. doi: 10.1042/bj2840891. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de la Peña P., Delgado L. M., del Camino D., Barros F. Two isoforms of the thyrotropin-releasing hormone receptor generated by alternative splicing have indistinguishable functional properties. J Biol Chem. 1992 Dec 25;267(36):25703–25708. [PubMed] [Google Scholar]