Reconstitution of the myometrial oxytocin receptor into proteoliposomes

Abstract

The requirements for regaining high-affinity binding of the myometrial oxytocin receptor after detergent solubilization were investigated by reconstitution experiments. Large unilamellar liposomes were prepared by reverse-phase evaporation from different mixtures of phospholipids, cholesterol and cholesteryl hemisuccinate. In the presence of the oxytocin receptor solubilized from myometrial membranes from pregnant guinea pig uterus, liposomes were treated with 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-l-propanesulfonate (Chapso) throughout the range of detergent concentrations that cause the transformation of lamellar structures to mixed micelles. Detergent removal was achieved using bio-beads SM-2 as adsorbent. The presence of cholesterol was a prerequisite for regaining high-affinity binding of ['H]oxytocin and 'Z51-oxytocin antagonist to reconstituted proteoliposomes. Binding of ['Hloxytocin but not of the antagonist was dependent on the presence of Mn'+ ions. Reconstitution after lectin chromatography and photoaffinity labeling of reconstituted vesicles resulted in the exclusive labeling of the oxytocin receptor with a molecular mass of 68 -80 kDa.The actions of the neurohypophyseal nonapeptide oxytocin initially described and as yet best established are its contractile effects on uterine smooth muscle (Dale, 1906) and myoepithelial cells (Ott and Scott, 1910). Oxytocin is believed to stimulate contractility by increasing intracellular calcium levels (Carsten and Miller, 1985) as a result of receptor-mediated hydrolysis of phosphoinositol lipids (Marc et al., 1986).Recently, the primary structure of the human oxytocin receptor from uterine myometrium was elucidated after its cDNA had been isolated by expression cloning (Kimura et al., 1992). The encoded oxytocin receptor is a 389-aminoacid polypeptide with seven hydrophobic putative transmembrane domains typical for guanine-nucleotide-binding protein (G-protein) coupled receptors. The protein core of the receptor has an apparent molecular mass of 42.8 kDa. Protein chemical studies, especially photoaffinity labeling experiments and enzymic deglycosylation of the myometrial oxytocin receptor from guinea pig, showed that the oxytocin receptor is highly glycosylated, with an apparent molecular mass between 68-80kDa (Kojro et al., 1991). At present, the functional domains of the receptor protein responsible for hormone binding and coupling with G-proteins are unknown, mainly because the purification of the receptor protein has not been achieved.Guinea pig uterus is the tissue of choice for purification of the myometrial oxytocin receptor because of its high receptor density at late pregnancy (Fahrenholz et al., 1988). In contrast to the solubilization of the oxytocin receptor from rat mammary gland (Soloff and Fernstrom, 1987), it has so far not been possible to achieve solubilization of the oxytocin receptor from guinea pig uterus without losing its binding activity.A role of phospholipids in the interaction of oxytocin with its receptor in mammary gland...

Glutamate-binding affinity ofDrosophilametabotropic glutamate receptor is modulated by association with lipid rafts

“…The oxytocin receptor is the only other GPCR for which it is known that ligand binding also depends on the presence of sterols (36)(37)(38). This receptor belongs to the rhodopsin-like GPCR family, which possesses a ligand-binding pocket in its transmembrane domain.…”

Regulation of receptor function by cholesterol

“…In the early 1990s, Klein and Fahrenholz [80] described reconstitution experiments with the oxytocin receptor (OTR) of guinea pig uterus. These experiments demonstrated that cholesterol is a prerequisite for the highaffinity binding activity (K d in the low nanomolar range) of the myometrial OTR after reconstitution.…”

Two Classes of Cholesterol Binding Sites for the β 2 AR Revealed by Thermostability and NMR

“…Effects of cholesterol on the stability and function of G protein-coupled receptors (GPCRs) have been known for at least two decades (1)(2)(3). However, there is often no clear consensus as to whether the effects of cholesterol on individual protein species arise from a specific GPCR-cholesterol interaction, from the effect of cholesterol on the bulk properties of the lipid bilayer, or from a combination of both (4).…”