Abstract
The human vasoactive intestinal peptide (VIP) 1 receptor belongs to the new class II subfamily of G protein-coupled receptors. Specific change by mutagenesis of a strictly conserved histidine into arginine at position 178 of the human VIP1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of the H178R mutant into COS cells resulted in a 3.5-fold increase in the cAMP level as compared with cells transfected with the wild type receptor or the vector alone. This increase was proportional to the amount of transfected cDNA. The H178R mutant exhibited an otherwise normal cAMP response to VIP as well as a dissociation constant similar to that of the wild type receptor. Other mutants at position 178 such as H178K, H178A, and H178D were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the H178R mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production, i.e. E36A or D68A. The corresponding double mutants H178R/E36A and H178R/D68A were no longer constitutively activated. A control double mutant (H178R/D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP was still constitutively activated. Our findings demonstrate that constitutive activation of the VIP1 receptor by mutation of His178 into R requires the functional integrity of the N-terminal extracellular VIP binding domain. They might provide interesting generalities about the activation process of G protein-coupled receptors.
Highlights
G protein-coupled receptors with seven transmembrane domains, which constitute a large multigene family of eucaryotic proteins, interact with alkaloids, biogenic amines, peptides, glycoprotein hormones, light, and odorants [1]
The histidine-to-arginine mutant (H178R) of the human VIP1 receptor, which mimics the situation found in constitutively activated parathyroid hormone (PTH) receptors [27], was transfected into monkey kidney COS-7 cells, and intracellular cAMP was measured
The concentration of vasoactive intestinal peptide (VIP) binding sites was higher in cells transfected with the wild type receptor than in cells transfected with the H178R receptor mutant (Table I)
Summary
Materials—Enzymes for cloning, sequencing, and oligonucleotidedirected mutagenesis were obtained from Promega (Madison, WI) or Life Technologies, Inc. (Cergy-Pontoise, France), and synthetic oligonucleotides were from Eurogentec (Seraing, Belgium). [␣-35S]dATP (1000 Ci/mmol) and other radioactive reagents were obtained from Amersham (Buckinghamshire, United Kingdom). The culture medium was discarded, and attached cells were gently rinsed with phosphate-buffered saline (pH 7) They were incubated with or without VIP under continuous agitation in 0.5 ml of phosphate-buffered saline containing 2% (w/v) bovine serum albumin, 0.1% (w/v) bacitracin, 0.01 mg/ml aprotinin, and 1 mM 3-isobutyl-1-methylxanthine as described [7]. Confocal Laser Scanning Microscopy—Transfected cells were grown on 12-mm glass coverslips for 48 h as described above After they were washed with phosphate-buffered saline (PBS), nonpermeabilized cells were incubated for 60 min at room temperature with the mouse monoclonal anti-Flag antibodies diluted 1:50 in PBS containing 1% (w/v) bovine serum albumin. Transfected cells grown in 24-well trays (see above) were rinsed twice with 50 mM Tris-HCl (pH 7.7), 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5% heat-inactivated horse serum, and 0.5% heat-inactivated fetal bovine serum (binding buffer), incubated for 4 h at room temperature with anti-Flag antibodies diluted 1:50 in binding buffer. Binding of anti-Flag antibodies to epitope-tagged mutant receptors was given as a percentage of anti-Flag antibodies binding to epitope-tagged wild type receptor
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