A Fusion Protein of the Human P2Y₁ Receptor and NTPDase1 Exhibits Functional Activities of the Native Receptor and Ectoenzyme and Reduced Signaling Responses to Endogenously Released Nucleotides

Abstract

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y 1 receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y 1 receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y 1 -NTPDase1 fusion protein. Stable expression of the P2Y 1 -NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca 2ϩ response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADP␤S to stimulate inositol phosphate accumulation was similar, and the EC 50 of ADP␤S was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y 1 and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y 1 receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y 1 receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y 1 receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

Regulation of P2Y1 Receptor-Mediated Signaling by the Ectonucleoside Triphosphate Diphosphohydrolase Isozymes NTPDase1 and NTPDase2

“…The inhibitory action of NTPDase1 was similar irrespective of whether the ectoenzyme was coexpressed on the same cell as the P2Y 1 receptor or was expressed on a different cell in cocultures with P2Y 1 receptor-expressing cells. Similar results were observed previously with a fusion protein of the human P2Y 1 receptor and NTPDase1 (Alvarado-Castillo et al, 2002). Addition of ATP or ADP to the bulk medium in these experiments may approximate the physiological situation in which activating nucleotide is released from a distant cell.…”

“…Heterologous expression of P2Y receptors has been widely reported to result in elevation of "basal" [ 3 H]inositol phosphate accumulation (Filtz et al, 1994;Parr et al, 1994;Lazarowski et al, 1995;Alvarado-Castillo et al, 2002). …”

Cellular function and molecular structure of ecto-nucleotidases

“…Using FRET microscopy of heterologously expressed fluorescence-tagged proteins, close molecular interaction, indicating complex formation, was observed between rat NTPDase1 and NTPDase2 and between NTPDase1 and a variety of P2Y nucleotide and P1 adenosine receptors [164]. The close interaction between NTPDases and P2Y receptors would have a severe impact on the availability of nucleotide agonists at their receptor, as has been implicated from the analysis of an NTPDase1/P2Y 1 receptor fusion protein [165].…”

The GDA1_CD39 superfamily: NTPDases with diverse functions

“…Because of the location of NTPDase1 on endothelial cells and its ability to hydrolyze ADP, a ligand of the P2Y1 receptor that promotes platelet aggregation, its important role in thromoboregulation was immediately recognized. Many in vitro studies followed that showed (1) an inverse correlation of NTPDase1 activity with platelet aggregation [28,66,67], (2) a decrease of NTPDase1 activity upon endothelial cell activation [67], and (3) attenuation of ATP signaling in cells expressing an NTPDase1-P2Y1 receptor fusion protein [68]. The last study demonstrated the importance of NTPDase1 in regulating purinergic signaling.…”