The angiotensin II binding site on Mycoplasma hyorhynis is structurally distinct from mammalian AT1 and AT2 receptors

Angiotensin II (AngII) binding sites were characterized on rat pheochromocytoma cells (PC-12) which are known to express exclusively the type-2 (AT(2)) AngII receptor. Interestingly, we found that, on confluent PC-12 cells, only partial inhibition of I-125-AngII binding was achieved when cells were incubated with a saturating concentration of PD-123 319 (an AT(2) selective ligand) suggesting the presence of an atypical binding site. In binding experiments, AngII exhibited high affinity for this atypical binding site with a dissociation constant (K-d) of 16 nM. Moreover, bacitracin potently inhibited PD-123 319-resistant I-125-AngII binding with an IC50 half-maximal inhibitory concentration of 44 mu M. Enzyme immunoassay revealed that the cells were contaminated with Mycoplasma hyorhynis. Contaminated PC-12 cells were photolabeled with I-125-[p-benzoylPhe(1)]AngII and covalently labeled proteins were subjected to polyacrylamide gel electrophoresis followed by autoradiography. Under these conditions, two distinct labeled species of 140 kilodaltons (kDa) and 95 kDa were detected. Deglycosylation of the 140 kDa-labeled AT(2) receptor with glycopeptidase-F (PNGase-F) resulted in a 35 kDa protein whereas the 95 kDa band was not affected by digestion with the endoglycosidase. Thus, our results show that the AngII binding site on M. hyorhynis is structurally distinct from mammalian AT(1) and AT(2) receptors. (C) 1998 Elsevier Science B.V.