The C-terminal third intracellular loop of the rat AT1A 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 (AT1A) 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 AT1A receptor (residues 234-240) was replaced with the homologous regions of the alpha1B adrenergic (alpha1B-AR), the beta2 adrenergic (beta2-AR), and the AngII type 2 (AT2) 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, [3H]thymidine incorporation into DNA, and internalization. The affinities of these chimeric receptors for [Sar1]AngII, [Sar1,Ile8]AngII, and losartan were essentially normal; however, the affinity of these mutants was increased by a factor of 10-40 for the AT2-specific ligand CGP42112A. The functional properties of the alpha1B-AR chimera were essentially identical to those of the wild type AT1A receptor. On the other hand, replacement with the beta2-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 AT2 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 AT1A 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.
Conchon
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INSERM Unité 36, Collège de France, 3, rue d'Ulm 75005 Paris, France.
Barrault
M B
MB
Miserey
S
S
Corvol
P
P
Clauser
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E
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004261:DNA Replication; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000096:biosynthesis; D008938:Mitosis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
D007295:Inositol Phosphates; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins