The C-terminal third intracellular loop of the rat AT(1A) angiotensin receptor plays a key role in G protein coupling specificity and transduction of the mitogenic signal

To identify the role(s) of the third intracellular loop of the angiotensin II (AngII) type 1A (AT(1A)) receptor in G protein coupling specificity and receptor activation, several chimerae were constructed and characterized, The cDNA sequence encoding the C-terminal segment of the third intracellular loop of the AT(1A) receptor (residues 234-240) was replaced with the homologous regions of the alpha(1B) adrenergic (alpha(1B)-AR), the beta(2) adrenergic (beta(2)-AR), and the AngII type 2 (AT(2)) receptors. These chimeric receptors were stably expressed in Chinese hamster ovary cells, and their pharmacological and functional properties were characterized, including AngII-induced inositol phosphate and cyclic AMP (cAMP) productions, [H-3]thymidine incorporation into DNA, and internalization. The affinities of these chimeric receptors for [Sar(1)]AngII, [Sar(1),Ile(8)]AngII, and losartan were essentially normal; however, the affinity of these mutants was increased by a factor of 10-40 for the AT(2)-specific ligand CGP42112A. The functional properties of the alpha(1B)-AR chimera were essentially identical to those of the wild type AT(1A) receptor. On the other hand, replacement with the beta(2)-AR segment produced a partial reduction of the inositol phosphate production, a measurable AngII-induced cAMP accumulation, a reduced internalization, and a total impairment to transduce the mitogenic effect of AngII. The AT(2) chimera presented a normal internalization, but was inactive in all the other functional tests. In conclusion, the distal segment of the third intracellular loop of the rat AT(1A) receptor plays a pivotal role in coupling selectivity and receptor signaling via G protein(s) as well as in the activation of the specific signaling pathways involved in the mitogenic actions of AngII.