Related Experiment Videos
Two basic residues of the h-VPAC1 receptor second transmembrane helix are essential for ligand binding and signal
R M Solano1, I Langer, J Perret
1Laboratoire de Chimie Biologique et de la Nutrition, Faculté de Médecine, Université Libre de Bruxelles, 808 route de Lennik, Building G/E, CP 611, B-1070 Brussels, Belgium.
Abstract:
We mutated the vasoactive intestinal peptide (VIP) Asp(3) residue and two VPAC(1) receptor second transmembrane helix basic residues (Arg(188) and Lys(195)). VIP had a lower affinity for R188Q, R188L, K195Q, and K195I VPAC(1) receptors than for VPAC(1) receptors. [Asn(3)] VIP and [Gln(3)] VIP had lower affinities than VIP for VPAC(1) receptors but higher affinities for the mutant receptors; the two basic amino acids facilitated the introduction of the negatively charged aspartate inside the transmembrane domain. The resulting interaction was necessary for receptor activation. 1/[Asn(3)] VIP and [Gln(3)] VIP were partial agonists at VPAC(1) receptors; 2/VIP did not fully activate the K195Q, K195I, R188Q, and R188L VPAC(1) receptors; a VIP analogue ([Arg(16)] VIP) was more efficient than VIP at the four mutated receptors; and [Asn(3)] VIP and [Gln(3)] VIP were more efficient than VIP at the R188Q and R188L VPAC(1) receptors; 3/the [Asp(3)] negative charge did not contribute to the recognition of the VIP(1) antagonist, [AcHis(1),D-Phe(2),Lys(15),Arg(16),Leu(27)] VIP ()/growth hormone releasing factor (8-27). This is the first demonstration that, to activate the VPAC(1) receptor, the Asp(3) side chain of VIP must penetrate within the transmembrane domain, in close proximity to two highly conserved basic amino acids from transmembrane 2.
Insights
This study shows that the Asp(3) residue of vasoactive intestinal peptide (VIP) must interact with basic residues in the VPAC(1) receptor for activation. This interaction is crucial for receptor signaling and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Vasoactive intestinal peptide (VIP) is a key signaling molecule.
- VPAC(1) receptors mediate VIP's biological effects.
- Understanding receptor-ligand interactions is vital for drug development.
Purpose of the Study:
- To investigate the role of VIP's Asp(3) residue and VPAC(1) receptor transmembrane basic residues in receptor activation.
- To elucidate the mechanism of VPAC(1) receptor signaling.
Main Methods:
- Site-directed mutagenesis of VIP and VPAC(1) receptor.
- Ligand binding assays to determine receptor affinity.
- Functional assays to assess receptor activation and agonist efficacy.
Main Results:
- Mutating VPAC(1) receptor basic residues (Arg(188), Lys(195)) reduced VIP affinity.
- VIP Asp(3) mutations ([Asn(3)] VIP, [Gln(3)] VIP) showed altered affinities and partial agonism.
- The interaction between VIP Asp(3) and VPAC(1) basic residues is necessary for full receptor activation.
Conclusions:
- VIP Asp(3) side chain penetration into the transmembrane domain is essential for VPAC(1) receptor activation.
- This interaction with conserved basic residues in transmembrane 2 is a critical step in VIP signaling.
- Findings provide insights into GPCR activation mechanisms.