Characterization of adenosine binding proteins in human placental membranes.

Abstract

We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with ($\sp3$H) -N-ethylcarboxamidoadenosine ( ($\sp3$H) NECA). This site is similar to the adenosine A$\sb2$ receptor. We call this site the adenosine A$\sb2$-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A$\sb1$ receptor. The soluble adenosine A$\sb2$-like binding site cannot be detected without a rapid assay. Binding to the adenosine A$\sb1$ receptor with ($\sp3$H) -2-chloroadenosine and ($\sp3$H) NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A$\sb1$ specificity: 2-chloroadenosine $>$ R-phenylisopropyladenosine $>$ 5$\sp\prime$-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine $>$ isobutylmethylxanthine $>$ theophylline. Competition analysis of membranes with the A$\sb1$-selective ligands ($\sp3$H) -cyclohexyladenosine and ($\sp3$H) -cyclopentylxanthine revealed adenosine A$\sb1$ agonist and antagonist potency orders. We have purified the adenosine A$\sb2$-like binding site. The adenosine A$\sb2$-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 A. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A$\sb2$-like binding site has an agonist potency order of 5$\sp\prime$-N-ethylcarboxamidoadenosine $>$ 2-chloroadenosine $\gg$ R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine $>$ theophylline $\gg$ 1,3-diethyl-8-phenylxanthine. The adenosine A$\sb2$-like binding site is present in membranes and the 105,000 x g supernatant of placental homogenates. The membrane and soluble forms are identical by amino terminal sequence, amino acid composition, and subunit molecular mass. The membrane site differs from the soluble site by a decreased negative charge and an increased weight % carbohydrate. We present an hypothesis relating the membrane, soluble, and a proteolyzed form (74 kDa subunit molecular mass) of the soluble adenosine A$\sb2$-like binding site. The amino terminus of the adenosine A$\sb2$-like binding site is highly homologous with several vertebrate stress-regulated proteins: hamster GRP94, murine ERP99 and GP96, and chicken HSP108. By homology with these stress-regulated proteins, the adenosine A$\sb2$-like binding site may be a human heat-shock protein.