INTRACELLULAR 3RD LOOP DOMAIN OF ANGIOTENSIN-II TYPE-2 RECEPTOR - ROLE IN MEDIATING SIGNAL-TRANSDUCTION AND CELLULAR FUNCTION

The present study tests the hypothesis that the unique intracellular t hird loop domain of angiotensin II type-2 (AT2) receptor is essential for the subsequent intracellular signaling and plays an important role in mediating receptor function, Synthetic intracellular third loop pe ptide of the AT2 receptor (AT2-3LP, 22 amino acids) and control peptid e consisting of the same amino acid composition in random sequence wer e delivered into adult rat aortic vascular smooth muscle cells by cati onic liposome-mediated transfection. Successful intracellular peptide delivery was confirmed by microscopic localization of the fluorescein- labeled AT2-3LP within the cells and also by co-immunoprecipitation of the I-125-labeled 3LP complexed with G(i) protein using anti-G(i) alp ha antibody. The AT2-3LP-transfected cells showed reduction of serum-s timulated DNA synthesis and cell proliferation as well as a decrease i n mitogen-activated protein kinase activity, simulating the effects of AT2 receptor stimulation. The antagonistic effect of the AT2-3LP on m itogen-activated protein kinase activity and DNA synthesis were revers ed by pertussis toxin and sodium orthovanadate. Thus, our data suggest that the intracellular third loop domain of the AT2 receptor is close ly linked with the cellular signaling pathways of vascular smooth musc le cells in which G(i) and protein-phosphotyrosine phosphatase are inv olved, resulting in the alteration of mitogen-activated protein kinase activity and in growth inhibition.