Production and characterization of multiple antigenic peptide antibodies to the adenosine A2b receptor

A polyclonal antibody to the human adenosine A2b receptor (A2bR) was produced by immunizing a chicken with a multiple antigenic peptide consisting of eight copies of a 16-amino acid peptide, corresponding to the presumed second extracellular loop of the A2bR, linked to a branched lysine core. Western blotting with affinity-purified antibody revealed the human A2bR to be a protein of approximately 50-55 kDa, found in a variety of tissues including thymus, colon, and small intestine. The antibody also recognized mouse and rat A2bRs and revealed heterogeneity in size, with a 35-kDa protein being detected in small intestine in addition to the larger 50-52-kDa species in thymus, colon, and placenta. The chicken anti-human A2bR peptide antibody recognized the receptor in both frozen and formalin-fixed tissue sections. In human colon, the A2bR was highly expressed in epithelial cells of the crypts. A2bR immunoreactivity was also apparent in syncytiotrophoblast cells of human placental villi and in the basal zone of murine chorioallantoic placenta. These cell type-specific patterns of expression are consistent with the hypothesized roles of the A2bR in mediating electrogenic Cl- secretion and the resulting secretory diarrhea caused by colonic crypt abscesses and in regulating morphogenesis of the placenta. Insight into the multiple physiological consequences of A2bR engagement will be forthcoming from an analysis of the cell type-specific expression of this receptor in additional tissues.