Identification of the region of AT2 receptor needed for inhibition of the AT1 receptor-mediated inositol 1,4,5-triphosphate generation.
Increase in the intracellular inositol triphosphate (IP3) levels in Xenopus oocytes in response to expression and activation of rat angiotensin II (Ang II) receptor AT1 was inhibited by co-expression of rat AT2 receptor. To identify which region of the AT2 was involved in this inhibition, ability of three AT2 mutants to abolish this inhibition was analyzed. Deletion of the C-terminus of the AT2 did not abolish this inhibition. Replacing Ile249 in the third intracellular loop (3rd ICL) of the AT2 with proline, corresponding amino acid in the AT1, in the mutant M6, resulted in slightly reduced affinity to [125I]Ang II (K(d)=0.259 nM), however, did not abolish the inhibition. In contrast, replacing eight more amino acids in the 3rd ICL of the AT2 (at positions 241-244, 250-251 and 255-256) with that of the AT1 in the mutant M8, not only increased the affinity of the AT2 receptor to [125I]Ang II (K(d)=0.038 nM) but also abolished AT2-mediated inhibition. Interestingly, activation of the M8 by Ang II binding also resulted in increase in the intracellular IP(3) levels in oocytes. These results imply that the region of the 3rd ICL of AT2 spanning amino acids 241-256 is sufficient for the AT2-mediated inhibition of AT1-stimulated IP3 generation. Moreover, these nine mutations are also sufficient to render the AT2 with the ability to activate phospholipase C.
Kumar
Vikas
V
Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
Knowle
Dieter
D
Gavini
Narasaiah
N
Pulakat
Lakshmidevi
L
D000595:Amino Acid Sequence; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D010738:Type C Phospholipases / Q000378:metabolism; D014981:Xenopus
D008024:Ligands; D018075:RNA, Complementary; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D007532:Isoleucine; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases