Analysis of the C-terminal tail of the rat thyrotropin-releasing hormone receptor-1 in interactions and cointernalization with beta-arrestin 1-green fluorescent protein.
Coexpression of the rat thyrotropin releasing hormone receptor-1 with beta-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 beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-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 beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.
Groarke
D A
DA
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow Scotland, United Kingdom.
Drmota
T
T
Bahia
D S
DS
Evans
N A
NA
Wilson
S
S
Milligan
G
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; C000605256:Arrb1 protein, rat; D019390:Arrestins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018025:Receptors, Thyrotropin-Releasing Hormone; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D002118:Calcium