The Structural Determinants of GPCR Binding and Activations; Insights from Mutagenesis and Molecular Modeling Studies of the Human Angiotensin II Type 1 (hATl) Receptor

摘要

The G protein coupled receptors (GPCRs) are seven transmembrane domains (TMD) proteins involved in the mediation of environmental stimuli across the plasma membrane. Determination of the structural basis of GPCR ligand binding and activation is of primary importance in drug development. However, their structural analysis remains a considerable challenge and the only GPCR 3D structure experimentally determined to date is that of the bovine rhodopsin. The mutation of an Asn residue in TMD 3, conserved in 29% of all family A GPCRs, has been reported to induce constitutive activation of several GPCRs, such as the PAF, B2, CXCR4, and hATl receptors. Rhodopsin-based homology models of the hAT1 receptor revealed an interaction between this residue (N111~(3.35), Ballesteros numerotation in superscript) and a conserved Asp residue in TMD 2, D74(2.50) (Fig. 1A). Interestingly, mutation of D74 impairs isomerization of the receptor toward an active form capable of activating the G_q protein. We hypothesized that the interaction between D74 and Nl 11 is crucial for the stabilization of the inactive form of hATl in the absence of agonist and that this interaction maintains the GELTA G of isomerisation (DELTA G_(iso)) within a few kcal/mol in order to allow a small population of receptors to adopt the active form. We generated D74N and N1 11G mutant hATl receptors as controls, as well as two novel mutant receptors, D74N/N111D and D74N/N111G. After verifying the structural integrity of the mutant receptors by saturation and competition studies with angiotensin II (Ang II), their ability to activate G_q was assessed by IP production analysis (Fig. 1B).