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.
Mesh Terms:
Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Carrier Proteins, Cell Membrane, Cells, Cultured, Coatomer Protein, Eukaryotic Cells, GTP-Binding Proteins, Green Fluorescent Proteins, Humans, Indicators and Reagents, Intracellular Membranes, Luminescent Proteins, Mutagenesis, Mutation, Protein Binding, Protein Structure, Tertiary, Protein Transport, Rats, Receptors, Cell Surface, Receptors, Dopamine D1, Two-Hybrid System Techniques, beta-Galactosidase
Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Carrier Proteins, Cell Membrane, Cells, Cultured, Coatomer Protein, Eukaryotic Cells, GTP-Binding Proteins, Green Fluorescent Proteins, Humans, Indicators and Reagents, Intracellular Membranes, Luminescent Proteins, Mutagenesis, Mutation, Protein Binding, Protein Structure, Tertiary, Protein Transport, Rats, Receptors, Cell Surface, Receptors, Dopamine D1, Two-Hybrid System Techniques, beta-Galactosidase
Eur. J. Cell Biol.
Date: Feb. 01, 2002
PubMed ID: 11893085
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