Vol. 62, Issue 3, 521-528, September 2002

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

Claudia Alvarado-Castillo, Patricia Lozano-Zarain, Jesús Mateo, T. Kendall Harden, and José L. Boyer

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (C.A.-C., P.L.-Z., J.M., T.K.H., J.L.B.); and Inspire Pharmaceuticals, Inc., Durham, North Carolina (J.L.B.)

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y₁ 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 P2Y₁ 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 P2Y₁-NTPDase1 fusion protein. Stable expression of the P2Y₁-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca²⁺ response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPS to stimulate inositol phosphate accumulation was similar, and the EC₅₀ of ADPS 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 P2Y₁ 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 P2Y₁ receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y₁ receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y₁ receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.