No abstract

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto-enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADMCD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A 1 adenosine receptor (AIR) is a second ecto-ADA binding protein. Binding of ADA to AtR increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A~R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand-induced second messenger production via AtR. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto-adenosine deaminase on the cell surface is to facilitate the signal transduction via AIR.

SUMMARYThrough immunocytochemistry with the use of antibodies against A 1 adenosine receptors (A 1 Rs) and confocal microscopy, we show that stimulation of A 1 Rs by the agonist (R)-phenylisopropyladenosine [(R)-PIA] caused a rapid (5-15 min) aggregation (clustering) of receptor molecules on the surface of DDT 1 MF-2 cells. Internalization of the chronically stimulated receptor was slower and occurred concomitantly, with a timedependent decrease (50%) in the number of cell surface [ 3 H](R)-PIA binding sites. The reduction of binding sites was due partly (30%) to internalization and partly (20%) to the presence of desensitized cell surface receptor molecules that were unable to bind the ligand. Chronic exposure of DDT 1 MF-2 cells to 50 nM (R)-PIA produced functional desensitization, as deduced from second messenger production assays. Quantification of the content of A 1 Rs by immunoblotting and flow cytometry in cells pretreated with 50 nM (R)-PIA indicates a time-dependent slow down-regulation of the receptor. Receptor clustering and agonist-induced receptor phosphorylation, which occurred in serine and tyrosine, were simultaneous. The finding that activators of protein kinase A or C were able to induce functional desensitization of A 1 Rs, phosphorylate A 1 Rs in serine and threonine, and trigger clustering of the receptor suggests that phosphorylation of A 1 Rs in serine/threonine is involved in desensitization-related events.

Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A1 adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high‐affinity site of the receptor, which corresponds to the receptor‐G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A1 adenosine receptors to heterotrimeric G proteins.

Adenosine deaminase (ADA, E.C. 3.5.4.4) is an enzyme of the purine metabolism that converts adenosine (Ado) and 2′deoxyadenosine (dAdo) into inosine and 2′deoxyinosine, respectively. The enzyme has been the object of considerable interest because the congenital defect causes severe combined immunodeficiency (SCID). In the past 12 years, ADA, which was considered to be cytosolic, has been found on the surface of many cells; therefore, it has to be also considered as an ecto‐enzyme (ecto‐ADA). There is recent evidence about a specific role for ecto‐ADA that is different from that of intracellular ADA. Apart from degrading extracellular Ado or dAdo, which are toxic for lymphocytes and other cells, ecto‐ADA has got an extraenzymatic function by means of its interaction with cell surface proteins. The interactions in which ecto‐ADA participates are summarized, and an overview of the known and suspected extraenzymatic roles for ecto‐ADA is presented. Drug Dev. Res. 45:261–268, 1998. © 1998 Wiley‐Liss, Inc.