Polar residues play essential roles in the functions of transmembrane helices by mediating and stabilizing their helical interactions. To investigate the structural/functional roles of the conserved polar residues in the N-and C-terminal transmembrane helices of human NTPDase3 (N-terminal: S33, S39, T41, and Q44; C-terminal: T490, T495, and C501), each was singly mutated to alanine. The mutant proteins were analyzed for enzymatic activities, glycosylation status, expression level, and Triton X-100 detergent sensitivity. The Q44A mutation decreased Mg-ATPase activity by approximately 70%, and abolished Triton X-100 detergent inhibition of Ca-dependent nucleotidase activities, while greatly attenuating Triton X-100 inhibition of Mg-dependent nucleotidase activities. The polar residues were also mutated to cysteine, singly and in pairs, to allow a disulfide cross-linking strategy to map potential inter-and intra-molecular hydrogen bond interactions. The results support the centrality of Q44 for the strong intermolecular interactions driving the association of the Nterminal helices of two NTPDase3 monomers in a dimer, and that T41 may play a role in specificity of this interaction. In addition, S33 and C501 form an intra-molecular association, while S39 and T495 may contribute to helical interactions involved in forming higher order oligomers. Lastly, Tween 20 substantially and selectively increases NTPDase3 activity, mediated by the transmembrane helices containing the conserved polar residues. Taken together, the data suggest a model for putative hydrogen bond interactions of the conserved polar residues in the transmembrane domain of native, oligomeric NTPDase3. These interactions are important for proper protein expression, full enzymatic activity, and susceptibility to membrane perturbations. Keywordsecto-nucleotidase; NTPDase3; transmembrane hydrogen bonds; conserved polar residues; disulfide cross-linking; Tween 20 stimulation; Triton X-100 inactivation Human nucleoside triphosphate diphosphohydrolase 3 (human NTPDase3) is a member of a family of ecto-enzymes that are characterized by their ability to hydrolyze extracellular and intra-luminal nucleoside di-and tri-phosphates in the presence of divalent cations (1). There are eight members in the human family of NTPDases. NTPDase1-3 and 8 are expressed on the cell-surface and hydrolyze extracellular nucleotides, while NTPDase4-7 hydrolyzes intraluminal nucleotides (2,3). NTPDase1-4, 7, and 8 are membrane-bound glycoproteins with a transmembrane (TM) helix near both their N-and C-termini, and a large extracellular domain containing five conserved disulfide bonds and the enzyme active site (4). NTPDase5 and 6 are Address correspondence to: Terence L. Kirley, Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, P.O. Box 670575, Cincinnati, OH 45267-0575. Phone: 513-558-2353; Fax: 513-558-9969; terry.kirley@uc.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 October 13....

The active sites of the membrane-bound nucleoside triphosphate diphosphohydrolases (NTPDases) regulate and are regulated by coordinated and spatially distant movements of their transmembrane helices, modulating enzyme activity, and substrate specificity. Using sitedirected mutagenesis, the roles of the conserved proline residues (N-terminal: P52 and P53; C-terminal: P472, P476, P481, P484, and P485) of human NTPDase3, located in the "linker regions" that connect the N-and C-terminal transmembrane helices with the extracellular active site, were examined. Single cysteine substitutions were strategically placed in the transmembrane domain (N-terminal helix: V42C; C-terminal helix: G489C) to serve as crosslinking "sensors" of helical interactions. These "sensor" background mutant proteins (V42C and G489C NTPDase3) are enzymatically active and are cross-linked by copper phenanthroline less efficiently in the presence of adenosine triphosphate (ATP). Proline to alanine substitutions at P53, P481, P484, and P485 in the V42C background, as well as P53, P481, and P484 in the G489C background, exhibited decreased nucleotidase activities. More importantly, alanine substitutions at P53 and P481 in the V42C background and P481 in the G489C background no longer exhibited the ATP-induced decrease in transmembrane cross-linking efficiency. Interestingly, the P485A mutation abolished oxidative cross-linking at G489C both in the presence and absence of ATP. Taken together, these results suggest a role for proline residues 53 and 481 in the linker regions of human NTPDase3 for coupling nucleotide binding at the enzyme active site to movements and/or rearrangements of the transmembrane helices necessary for optimal nucleotide hydrolysis.

Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) is a cell surface, membrane-bound enzyme that hydrolyzes extracellular nucleotides, thereby modulating purinergic signaling. An alternatively spliced variant of NTPDase3 was obtained and analyzed. This alternatively spliced variant, termed "NTPDase3β", is produced through the use of an alternative terminal exon (exon 11) in place of the terminal exon (exon 12) in the full-length NTPDase3, now termed "NTPDase3α". This results in an expressed protein lacking the C-terminal cytoplasmic sequence, the C-terminal transmembrane helix, and apyrase conserved region 5. The cDNA encoding this truncated splice variant was detected in a human lung library by PCR. Like the full-length NTPDase3α, the alternatively spliced NTPDase3β was expressed in COS cells after transfection, but only the fulllength NTPDase3α is enzymatically active and properly trafficked to the plasma membrane. However, when the truncated NTPDase3β was co-transfected with full-length NTPDase3α, there was a significant reduction in the amount of NTPDase3α that was properly processed and trafficked to the plasma membrane as active enzyme, indicating that the truncated form interferes with normal biosynthetic processing of the full-length enzyme. This suggests a role for the NTPDase3β variant in the regulation of NTPDase3 nucleotidase activity, and therefore the control of purinergic signaling, in those cells and tissues expressing both NTPDase3α and NTPDase3β.