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Tryptophan 67 in the human VPAC(1) receptor: crucial role for VIP binding
P Nicole1, J J Maoret, A Couvineau
1Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, Paris, 75018, France.
Tryptophan 67 (W67) in the VPAC(1) receptor is crucial for VIP binding. Mutating W67 significantly impairs or abolishes VIP binding and adenylyl cyclase activation, highlighting its importance.
Area of Science:
- Molecular Biology
- Biochemistry
- Receptor Pharmacology
Background:
- The vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor type 1 (VPAC(1) receptor) features a large N-terminal extracellular domain essential for VIP binding.
- Investigating specific amino acid residues within this domain can elucidate critical interactions for receptor function.
Purpose of the Study:
- To determine the role of specific amino acid residues, particularly tryptophan 67 (W67), in the N-terminal extracellular domain of the human VPAC(1) receptor for VIP binding and activation.
- To elucidate the structural contribution of W67 to the receptor's interaction with VIP.
Main Methods:
- Site-directed mutagenesis was used to alter 12 amino acid residues in the VPAC(1) receptor N-terminal domain, including W67, to alanine.
- Mutant cDNAs were transiently transfected into Cos cells and stably into CHO cells for expression analysis.
- Radioligand binding assays with (125)I-VIP and adenylyl cyclase activity assays were performed to assess receptor function.
- Cell surface expression of mutants was confirmed using microscopy and antibody binding.
Main Results:
- Most mutants exhibited binding affinities (K(d) values) similar to the wild-type receptor.
- The W67A mutant showed no specific (125)I-VIP binding.
- Mutations at W67 (W67A, W67E, W67H, W67K) abolished VIP binding and adenylyl cyclase activation, while W67F showed reduced activation at high VIP concentrations.
- All tested mutants were expressed at comparable levels on the cell surface.
Conclusions:
- Tryptophan 67 is critically important for VIP binding to the human VPAC(1) receptor.
- The aromatic nature of W67 likely contributes significantly to its role in mediating VIP binding.
- These findings provide insight into the structural basis of VIP receptor-ligand interactions.
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