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

Abstract

The angiotensin 11 receptors of human myometrial tissue were characterized using ligand binding, cross-linking with radioactive label, detergent solubilization and partial purification by lectinaffinity chromatography. Human myometrial membrane preparations contained variable amounts (5-650 fmol/mg protein) of high affinity (Kd = 44-65 pM) binding sites for '251-CGP42112, a ligand specific for the AT2 subtype of angiotensin I1 receptors. Competition studies with AT1-specific and AT2-specific compounds indicated that angiotensin I1 receptors on these membranes were exclusively of the AT2 subtype. The binding sites for '251-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 '251-[Sarl, Ile8langiotensin I1 (Sarile) with disuccinimidyl suberate. When low concentrations of cross-linker were used, a single radiolabelled band of about 66-70 kDa was revealed on SDSPAGE. 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 SDSPAGE 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 1251-Sarile-labelled protein with N-glycanase caused a shift in its apparent molecular mass on SDSPAGE from 66-70kDa to 33 kDa.The peptide hormone angiotensin I1 exerts its numerous physiological actions through binding to membrane receptors. At least two subtypes of receptors for angiotensin I1 exist, which can be distinguished on the basis of their different affinity for synthetic ligands. The non-peptide antagonist DuP753 (losartan) is specific for the AT1 receptor subtype, while the synthetic peptide CGP42112 is specific for the AT2 receptor subtype. The expression of these two subtypes is tissue-specific and species-specific ; some tissues express only one subtype while others express both [ 1, 21. Most known physiological actions of angiotensin I1 such as regulation of blood pressure, extracellular fluid volume, catecholamine and aldosterone secretion, etc., can be ascribed to its binding to the AT1 receptor [3, 41. Binding of angiotensin I1 to this subtype results in modulation of the CAMP and/or Ca2+ second-messenger pathways through activation of guanine-nucleotide-binding regulatory proteins (G proteins). Recently, the cDNA encoding AT1 receptors from several spe- Little is known about the AT2 receptor subtype, but apparently it also comprises several members with different molecular properties. Bovine cerebellum [ 111, human myometrium [ll -131, rat ovarian granulosa cells [14], the R3T3 [15]and PC12 W 1161 cell lines express AT2 recept...

Translocation of AT1- and AT2-Receptors by Higher Concentrations of Angiotensin II in the Smooth Muscle Cells of Rat Internal Anal Sphincter

“…Our studies demonstrate specific increase in the relative density of 105-to 120-kDa bands for AT 2 -R in the particulate fraction of the SMC with high concentrations of Ang II. In agreement with this concept, earlier studies in the human myometrium SMC have reported a similar increase in AT 2 -R homodimers in the PM (Lazard et al, 1994). RT-PCR analyses further show that relaxation of the SMC with higher concentrations of Ang II is associated with the cross-translocation of AT 1 -R and AT 2 -R rather than an overall change in their population density (Fig.…”

“…5B) without a significant change in the density of 50 to 70 kDa AT 2 -R in the particulate fraction. However, there was a significant increase in the density of 105 to 120 kDa AT 2 -R. The latter represents a homodimer of AT 2 -R (two units physically bound) as has already been shown in previous studies in different systems (Lazard et al, 1994).…”

Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats

“…AT 2 R expression corresponded to a band of molecular size 65 kDa (Fig. 1a), probably representing the glycosylated form of the receptor, as described previously [22,23].…”

Characterization of a Novel Iodocyanopindolol and SM-11044 Binding Protein, Which May Mediate Relaxation of Depolarized Rat Colon Tonus

“…Such absence was also reported for human and rat ␣2B-AR (29 -31). The molecular mass of human angiotensin II type 2 receptor was reported as 33 kDa after deglycosylation of 66 -70-kDa protein (32).…”