Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium

Eur J Biochem. 1994 Mar 15;220(3):919-26. doi: 10.1111/j.1432-1033.1994.tb18695.x.


The angiotensin II receptors of human myometrial tissue were characterized using ligand binding, cross-linking with radioactive label, detergent solubilization and partial purification by lectin-affinity chromatography. Human myometrial membrane preparations contained variable amount (5-650 fmol/mg protein) of high affinity (Kd = 44-65 pM) binding sites for 125I-CGP42112, a ligand specific for the AT2 subtype of angiotensin II receptors. Competition studies with AT1-specific and AT2-specific compounds indicated that angiotensin II receptors on these membranes were exclusively of the AT2 subtype. The binding sites for 125I-CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (Kd = 1.2 nM). The proteins in the myometrial membrane preparation were cross-linked to 125I-[Sar1, Ile8]angiotensin II (Sarile) with disuccinimidyl suberate. When low concentrations of cross-linker were used, a single radiolabelled band of about 66-70 kDa was revealed on SDS/PAGE. At higher concentrations additional bands of about 105-120 kDa and 200 kDa were labelled. The 66-70-kDa 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 bands are homodimers and trimers of the labelled polypeptide. The Chaps-solubilized receptor was retained on wheat-germ-agglutinin-Sepharose and specifically eluted by the competing sugar triacetylchitotriose, leading to a fivefold purification factor. Treatment of the 125I-Sarile-labelled protein with N-glycanase caused a shift in its apparent molecular mass on SDS/PAGE from 66-70 kDa to 33 kDa.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Sarcosine-8-Isoleucine Angiotensin II / metabolism
  • Cell Membrane / metabolism
  • Cross-Linking Reagents
  • Female
  • Humans
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / metabolism
  • Molecular Weight
  • Myometrium / chemistry*
  • Oligopeptides / metabolism
  • Receptors, Angiotensin / chemistry*
  • Receptors, Angiotensin / metabolism
  • Solubility


  • Cross-Linking Reagents
  • Membrane Glycoproteins
  • Oligopeptides
  • Receptors, Angiotensin
  • CGP 42112A
  • 1-Sarcosine-8-Isoleucine Angiotensin II