Analysis of the C-Terminal Tail of the Rat Thyrotropin-Releasing Hormone Receptor-1 in Interactions and Cointernalization with β-Arrestin 1-Green Fluorescent Protein
- D. Alex Groarke1,
- Tomas Drmota1,1,
- Daljit S. Bahia1,
- Nicholas A. Evans2,
- Shelagh Wilson2 and
- Graeme Milligan1
- 1Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow Scotland, United Kingdom (D.A.G., T.D., D.S.B., G.M.); and 2SmithKline Beecham Pharmaceuticals, Harlow, Essex, England, United Kingdom (N.A.E., S.W.)
Abstract
Coexpression of the rat thyrotropin releasing hormone receptor-1 with β-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the receptor. Truncations of the receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the receptor or its cointernalization with β-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with β-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with β-arrestin 1-GFP and a 10-fold reduction in internalization of the receptor. Despite this, the receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing hormone receptor-1 C-terminal tail required for effective contacts with β-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.
Footnotes
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Send reprint requests to: Dr. Graeme Milligan, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K. E-mail: g.milligan{at}bio.gla.ac.uk
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↵1 Current address: Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts.
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Financial support for this work was provided by the Biotechnology and Biosciences Research Council.
- Abbreviations:
- TRH
- thyrotropin-releasing hormone
- TRHR-1
- thyrotropin-releasing hormone receptor-1
- GFP
- green fluorescent protein
- HEK
- human embryonic kidney
- GPCR
- G protein-coupled receptor
- VSV
- vesicular stomatitis virus
- PCR
- polymerase chain reaction
- HEK
- human embryonic kidney cells
- DMEM
- Dulbecco's minimum essential medium
- RT
- room temperature
- PBSGG
- PBS containing 0.1% goat serum and 0.2% gelatin
- KRH
- Krebs-Ringer-HEPES
- FLIPR
- fluorometric imaging plate reader
- GnRH
- gonadotropin-releasing hormone
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- Received August 14, 2000.
- Accepted October 23, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
