ANGIOTENSIN-II RECEPTOR RECOGNIZED BY DUP753 REGULATES 2 DISTINCT GUANINE NUCLEOTIDE-BINDING PROTEIN SIGNALING PATHWAYS

The 7315c cell, derived from a rat anterior pituitary tumor, expresses an angiotensin II (AII) receptor. [H-3]AII binds to 7315c membranes s pecifically and saturably (K(D) = 2.1 +/- 0.6 x 10(-9) M, B(max) = 282 +/- 33 fmol/mg of protein). GTP diminished the affinity of the membra nes for [H-3]AII (K(D) = 4.1 +/- 0.4 x 10(-9) M, B(max) = 210 +/- 26 f mol/mg of protein). [H-3]AII binding was displaced by AII (K(i) = 1.3 +/- 0.6 x 10(-9) M, angiotensin III (AIII) (K(i) = 0.9 +/- 0.4 x 10(-9 ) M, and the nonpeptide AII antagonist DuP753 (K(i) = 1.4 +/- 0.6 x 10 (-8) M). In contrast, a second nonpeptide AII ligand, PD123177, did no t compete for [H-3]AII binding sites. In intact cells, AII and AIII st imulated inositol trisphosphate (IP3) production (EC50 = 1.1 +/- 0.6 x 10(-8) M and 1.1 +/- 0.5 x 10(-8) M, respectively); this response to AII was antagonized by DuP753 (K(i) = 1.7 +/- 0.3 x 10(-7) M). Pertuss is toxin treatment failed to affect the ability of AII to stimulate IP 3 production. In a crude membrane preparation, GTP was required for ma ximal AII-induced IP3 stimulation; guanosine thio-diphosphate abolishe d the agonist-GTP stimulation of IP3 production, in a concentration-de pendent fashion. AII and AIII also inhibited adenylyl cyclase (EC50 = 2.9 +/- 1.1 x 10(-8) M and 6.0 +/- 1.0 x 10(-8) M, respectively). DuP7 53 antagonized the inhibition by AII of adenylyl cyclase (K(i) = 2.8 /- 0.4 x 10(-8) M). PD123177 failed to antagonize AII-induced cyclase inhibition. Pertussis toxin treatment abolished the AII and AIII inhib ition of adenylyl cyclase. GTP was required for AII-induced inhibition of adenylyl cyclase. These data suggest that, in 7315c cells, a singl e subtype of AII receptor, identified by DuP753, is capable of regulat ing two different guanine nucleotide-binding protein (G protein) signa lling pathways; one G protein, which is insensitive to pertussis toxin , stimulates IP3 production and the other G protein, which is sensitiv e to pertussis toxin, inhibits adenylyl cyclase.