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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 site-directed 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 cross-linking "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.

Polar residues play essential roles in the functions of transmembrane helices by mediating and stabilizing their helical interactions. To investigate the structural and functional roles of the conserved polar residues in the N- and C-terminal transmembrane helices of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) (N-terminus, S33, S39, T41, and Q44; C-terminus, 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 intramolecular hydrogen bond interactions. The results support the centrality of Q44 for the strong intermolecular interactions driving the association of the N-terminal helices of two NTPDase3 monomers in a dimer, and the possibility that T41 may play a role in the specificity of this interaction. In addition, S33 and C501 form an intramolecular 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.

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 "NTPDase3beta", 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 "NTPDase3alpha". 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 NTPDase3alpha, the alternatively spliced NTPDase3beta was expressed in COS cells after transfection, but only the full-length NTPDase3alpha is enzymatically active and properly trafficked to the plasma membrane. However, when the truncated NTPDase3beta was co-transfected with full-length NTPDase3alpha, there was a significant reduction in the amount of NTPDase3alpha 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 NTPDase3beta variant in the regulation of NTPDase3 nucleotidase activity, and therefore the control of purinergic signaling, in those cells and tissues expressing both NTPDase3alpha and NTPDase3beta.