A fusion protein of the human P2Y(1) receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling responses to endogenously released nucleotides.
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(2+) response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC(50) of ADPbetaS 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.
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA. tkh@med.unc.edu
Claudia
C
Alvarado-Castillo
Patricia
P
Lozano-Zarain
Jesús
J
Mateo
T Kendall
TK
Harden
José L
JL
Boyer
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD; D001081:Apyrase / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D009711:Nucleotides / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D015703:Antigens, CD; D009711:Nucleotides; C546382:P2RY1 protein, human; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen