Interaction of gamma-COP with a transport motif in the D1 receptor C-terminus.
Truncations at the carboxyl termini of G protein-coupled receptors result in defective receptor biogenesis and comprise a number of inherited disorders. In order to evaluate the structural role of the C-terminus in G protein-coupled receptor biogenesis, we generated a series of deletion and substitution mutations in the dopamine D1 receptor and visualized receptor subcellular localization by fusion to a green fluorescent protein. Alanine substitutions of several hydrophobic residues within the proximal C-terminus resulted in receptor transport arrest in the ER. Agonist binding and coupling to adenylyl cyclase was also abolished. In contrast, substitutions conserving C-terminal hydrophobicity produced normal cell surface receptor expression, binding, and stimulatory function. A mechanism for the role of the C-terminus in D1 receptor transport was investigated by searching for candidate protein interactions. The D1 receptor was found to co-precipitate and associate in vitro directly with the gamma-subunit of the COPI coatomer complex. In vitro pull-down assays confirmed that only the D1 C-terminus is required for COPI association, and that identical mutations causing disruption of receptor transport to the cell surface also disrupted binding to COPI. Furthermore, conservative mutations in the D1 C-terminus restored COPI association just as they restored cell surface transport. These results suggest that association between the coatomer complex and hydrophobic residues within the proximal C-terminus of the D1 receptor may serve an important role in receptor transport.
Bermak
Jason C
JC
Department of Pharmacology, University of California, Irvine 92697, USA.
Li
Ming
M
Bullock
Clayton
C
Weingarten
Paul
P
Zhou
Qun-Yong
QY
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D020756:Coatomer Protein; D005057:Eukaryotic Cells / Q000378:metabolism* / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000096:biosynthesis*; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000235:genetics
D002352:Carrier Proteins; D020756:Coatomer Protein; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D017447:Receptors, Dopamine D1; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins