CHARACTERIZATION OF A MEMBRANE GLYCOPROTEIN HAVING PHARMACOLOGICAL AND BIOCHEMICAL-PROPERTIES OF AN AT2 ANGIOTENSIN-II RECEPTOR FROM HUMAN MYOMETRIUM

The angiotensin II receptors of human myometrial tissue were character ized using ligand binding, cross-linking with radioactive label, deter gent solubilization and partial purification by lectin-affinity chroma tography. Human myometrial membrane preparations contained variable am ounts (5-650 fmol/mg protein) of high affinity (K-d 44-65 pM) binding sites for I-125-CGP42112, a ligand specific for the AT2 subtype of ang iotensin II receptors. Competition studies with AT1-specific and AT2-s pecific compounds indicated that angiotensin II receptors on these mem branes were exclusively of the AT2 subtype. The binding sites for I-12 5-CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (K-d 1.2 nM). The proteins in the myometrial membrane preparation were cross linked to I-125-[Sar1, Ile8 ]angiotensin II (Sarile) with disuccinimidyl suberate. When low concen trations of cross-linker were used, a single radiolabelled band of abo ut 66-70 kDa was revealed on SDS/PAGE. At higher concentrations additi onal bands of about 105-120 kDa and 200 kDa were labelled. The 66-70-k Da and 105-120-kDa bands were separated by electroelution of SDS/PAGE gel slices and submitted to trypsin cleavage. The tryptic-peptide maps were identical for both products, suggesting that the additional band s are homodimers and trimers of the labelled polypeptide. The Chaps-so lubilized receptor was retained on wheat-germ-agglutinin-Sepharose and specifically eluted by the competing sugar triacetylchitotriose, lead ing to a fivefold purification factor. Treatment of the I-125-Sarile-l abelled protein with N-glycanase caused a shift in its apparent molecu lar mass on SDS/PAGE from 66-70 kDa to 33 kDa.