Structure of two fragments of the third cytoplasmic loop of the rat angiotensin II AT(1A) receptor: Implications with respect to receptor activation and G-protein selection and coupling

The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT(1A) receptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH₂-terminal half, Ni3, residues 213-231, adopts a stable amphipathic α-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH- terminal half, Ci3, residues 227-242. For this peptide, a cis-trans isomerization around the Lys⁶-Pro⁷ peptide bond generates two exchanging isomers adopting similar conformations, with an α-helix spanning from Asn⁹ to Ile¹⁵ and a poorly defined NH₂ terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic α-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro²³³ residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented.