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 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.
Lazard
D
D
ICGM, Laboratoire d'Immunopharmacologie Moléculaire, CNRS UPR 0415, Paris, France.
Villageois
P
P
Briend-Sutren
M M
MM
Cavaillé
F
F
Bottari
S
S
Strosberg
A D
AD
Nahmias
C
C
D015059:1-Sarcosine-8-Isoleucine Angiotensin II / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D005260:Female; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D009215:Myometrium / Q000737:chemistry*; D009842:Oligopeptides / Q000378:metabolism; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility
D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D011945:Receptors, Angiotensin; C060894:CGP 42112A; D015059:1-Sarcosine-8-Isoleucine Angiotensin II