Sylvia Y.M. Yao

The human concentrative (Na(+)-linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na(+)-nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691- and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na(+)-dependent cib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na(+) concentration indicated a Na(+):uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetate-induced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element.

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell in human and in characterization of its in the The protein to be a nucleoside transporter that to the nucleoside and and However, and the of the of the transporter in intracellular The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell in human and in characterization of its in the The protein to be a nucleoside transporter that to the nucleoside and and However, and the of the of the transporter in intracellular and transporters in the of in a of cell in particular in the and are the of hydrophilic and nucleoside and equilibrative nucleoside human mouse green fluorescent equilibrative nucleoside human mouse green fluorescent the of to cell transporters and in mammalian are of nucleoside transporter nucleoside found in and and members of the nucleoside transporter family, the family passive nucleoside are and are members of the equilibrative nucleoside transporter family, the family family widely distributed in its examples of of the members are to a of with a and and possess a and in the of the family hENT1 human and rodent designated The characterized of ENT1 and ENT2, are equilibrative nucleoside transporters that the of their to or The human differ in their to and with hENT1 the transporters from hENT1 in that in particular and a of and previously the of mouse and human ENT3 and a human the characterized a a transporter and designated plasma that the mouse in of contrast to the functional properties of hENT3 of the of a long hydrophilic N-terminal region which a targeting previously that hENT3 the the the the of the targeting motif the of hENT3 and that the transporter intracellular and with lysosomal of the hENT3 to the cell in the of the motif characterization of its functional properties in of hENT3 and previously the and of hENT3 and of the a region of and respectively, and the The the region of the of sequence and of a in which the dileucine motif and to alanine from the the N-terminal lysosomal targeting the region to of the from and respectively, and the The a sequence and The of of of a region of hENT3 and respectively, and The the region of hENT3 of a in which the dileucine motif and to alanine from the the N-terminal lysosomal targeting the region to of from and respectively, and the The a sequence and The of their in cultured the of hENT3 or of transporters were the green fluorescent protein The transporters with the to their of the and the of and were that the and of the hENT3 and the in in of and the of the were that with and the and of in were with of to the the were in with and in to of and of hENT3 and were in and previously cultured were in in with in and with in were with in and the of hENT3 were with that with a of The of endogenous hENT3 and of proteins were with mouse or or with with were with a or a of mouse the in in were in were with a to a and the of and hENT3 in hENT3 and were with and with in the of the of with and of the of and were previously that were to of and the to the and the and were the of hENT3 human with to of a and were in the were with the The to that human which a of the hENT3 and family members of of of hENT2, or in and The that the the hENT3 the and in the of hENT3 and human and mouse family designated The 475-residue hENT3 and proteins were found to be 73% identical in amino acid sequence and and to the human and mouse a in the of the amino acid sequence of the ENT3 proteins are in the and that of the ENT2, and ENT3 in of the from the mammalian to of the from of the human and mouse ENT3 the of the family and are to the the mammalian with a and However, differ from the mammalian in a typical (DE)XXXL(LI) endosomal/lysosomal targeting motif in the hydrophilic region of the sequence that the first motif its in ENT3 from and dileucine motif found the in the ENT3 from the from the of a lysosomal targeting motif that ENT3 intracellular a cell hENT3 were to the of the endogenous transporter in which to hENT3 of cell the a of with of the transporter protein of and and forms of the respectively, previously of of with a intracellular with cell The of the the in from of the with The of with that or markers with the endosomal/lysosomal with hENT3 a of intracellular with with or markers and with the lysosomal of hENT3 in and of endogenous were with and in the or of of the hENT3 were in and with from the in to The of hENT3 and of the transporter in hENT3 were markers of the the the or to the of the N-terminal dileucine motif in the of the a of the of proteins hENT3 hENT3 the first of the N-terminal region or hENT3 in which the dileucine motif and alanine contrast to the intracellular of in cell in or and intracellular in that with transcripts cell in with transcripts with of the N-terminal dileucine motif in the of of in proteins hENT3 hENT3 the first of the N-terminal region or hENT3 in which the dileucine motif and alanine to of in of with transcripts or or with and of hENT3 and of the of the ENT3 proteins the predominantly intracellular of hENT3 and its with the were in and the proteins and the intracellular targeting the of the and human and mouse proteins were that the of in were to in with the of mENT3, a hENT3 the of proteins, of were the intracellular targeting N-terminal of the protein or mutation of the of the motif to alanine and The the and identical the of the protein and the of the protein N-terminal The of the characterization of the of the the of in the of hENT3 a and contrast to of of the or the forms of in to in of and hENT3 in of in with the hENT3 transcripts or or in and or in and of of in with the transcripts or in and from to the of the transporter of and of which were the of the of and a of to of in The of the transporter of and and The in that hENT3 hENT1 and in a ENT2, the to ENT2, The of and the in and were and to and with respectively, of and The that and of of with transcripts or with and in of the of and of the of and and of in that in are of of in with the transcripts or in of and and of nucleoside and of a of nucleoside in The and were a of which from in its the in to a a of nucleoside the the that the and The and nucleoside in were the of a of with in the hENT1 the with a of and the and which of and with and of and that of were and with the of with of and of in with the transcripts or in were in in the or of the to of and the full-length of hENT3 and were respectively, from human and mouse of the that ENT3 widely in mouse and human tissues, and with a of the of ENT3 protein in mammalian of of region of identical to that of the protein and the are in The of in of the transporter protein with a particular abundance in and of and the to and forms of the respectively, were The of a from human and cell with to a of the hENT3 the of hENT3 transcripts in and The were found in a of tissues, and the were found in and of of from were and with The of proteins of are the and proteins are to be located the cell ENT1 in ENT1 and in the of cultured in the that the endogenous hENT3 proteins of cultured are predominantly located in intracellular with or of the transporter the cell of the of a of the human protein in in the intracellular of the protein and of cell targeting to the or the human and mouse ENT3 proteins, respectively, were in and found the lysosomal and hENT3 in the lysosomal and endogenous hENT3 hENT3 and the or that the transporter in part, in the of lysosomal previously a in membranes the of acid in the lysosomal to be the and in contrast to the equilibrative nucleoside transporter the ENT3 proteins of mouse, and a typical (DE)XXXL(LI) endosomal/lysosomal targeting motif The of motif in the intracellular targeting of ENT3 the of its to alanine or of the motif in its of the protein the human and mouse proteins, the of with transcripts the and the of of the dileucine motif alanine to in the of a of the transporter from the and cell to the plasma or mutation of the dileucine motif in which in of the transporter the a characterization to be of its and the transporter a from family members However, the to that of and to the of are to amino acid transporter of the of the the of the the the to the lysosomal of the of its of that the transporter and the respectively, and previously hENT1 the properties of nucleoside from are with the of of that in that to and the the plasma transporter hENT1 hENT1 and that the a of the of to of and and previously in the hENT1 to the of third that of the to be in nucleoside and characterized members of the family that to the plasma and be in the of from the lysosomal the transporters be to are with the nucleoside the lysosomal transporters. We to in the of the transporter to be or the of lysosomal transporters from of in lysosomal and acid transporter which and the of hENT3 to the and or of of in the transporters the which with nucleoside transporters and in the of and transporters in the of in a of cell in particular in the and are the of hydrophilic and nucleoside and equilibrative nucleoside human mouse green fluorescent equilibrative nucleoside human mouse green fluorescent the of to cell transporters and in mammalian are of nucleoside transporter nucleoside found in and and members of the nucleoside transporter family, the family passive nucleoside are and are members of the equilibrative nucleoside transporter family, the family family widely distributed in its examples of of the members are to a of with a and and possess a and in the of the family hENT1 The human and rodent designated The characterized of ENT1 and ENT2, are equilibrative nucleoside transporters that the of their to or The human differ in their to and with hENT1 the transporters from hENT1 in that in particular and a of and We previously the of mouse and human ENT3 and a human the characterized a a transporter and designated plasma that the mouse in of contrast to the functional properties of hENT3 of the of a long hydrophilic N-terminal region which a targeting previously that hENT3 the the the the of the targeting motif the of hENT3 and that the transporter intracellular and with lysosomal of the hENT3 to the cell in the of the motif characterization of its functional properties in of hENT3 and previously the and of hENT3 and of the a region of and respectively, and the The the region of the of sequence and of a in which the dileucine motif and to alanine from the the N-terminal lysosomal targeting the region to of the from and respectively, and the The a sequence and The of of of a region of hENT3 and respectively, and The the region of hENT3 of a in which the dileucine motif and to alanine from the the N-terminal lysosomal targeting the region to of from and respectively, and the The a sequence and The of their in cultured the of hENT3 or of transporters were the green fluorescent protein The transporters with the to their of the and the of and were that the and of the hENT3 and the in in of and the of the were that with and the and of in were with of to the the were in with and in to of and of hENT3 and were in and previously cultured were in in with in and with in were with in and the of hENT3 were with that with a of The of endogenous hENT3 and of proteins were with mouse or or with with were with a or a of mouse the in in were in were with a to a and the of and hENT3 in hENT3 and were with and with in the of the of with and of the of and were previously that were to of and the to the and the and were the of hENT3 human with to of a and were in the were with the The to that human which a of the hENT3 and family members of of of hENT2, or in and The that the the hENT3 the and in the of hENT3 and previously the and of hENT3 and of the a region of and respectively, and the The the region of the of sequence and of a in which the dileucine motif and to alanine from the the N-terminal lysosomal targeting the region to of the from and respectively, and the The a sequence and The of of of a region of hENT3 and respectively, and The the region of hENT3 of a in which the dileucine motif and to alanine from the the N-terminal lysosomal targeting the region to of from and respectively, and the The a sequence and The of their in cultured the of hENT3 or of transporters were the green fluorescent protein The transporters with the to their of the and the of and were that the and of the hENT3 and the in in of and the of the were that with and the and of in were with of to the the were in with and in to of and of hENT3 and were in and previously cultured were in in with in and with in were with in and the of hENT3 were with that with a of The of endogenous hENT3 and of proteins were with mouse or or with with were with a or a of mouse the in in were in were with a to a and the of and hENT3 in hENT3 and were with and with in the of the of with and of the of and were previously that were to of and the to the and the and were the of hENT3 human with to of a and were in the were with the The to that human which a of the hENT3 and family members of of of hENT2, or in and The that the the hENT3 the and in the of hENT3 and human and mouse family designated The 475-residue hENT3 and proteins were found to be 73% identical in amino acid sequence and and to the human and mouse a in the of the amino acid sequence of the ENT3 proteins are in the and that of the ENT2, and ENT3 in of the from the mammalian to of the from of the human and mouse ENT3 the of the family and are to the the mammalian with a and However, differ from the mammalian in a typical (DE)XXXL(LI) endosomal/lysosomal targeting motif in the hydrophilic region of the sequence that the first motif its in ENT3 from and dileucine motif found the in the ENT3 from the from the of a lysosomal targeting motif that ENT3 intracellular a cell hENT3 were to the of the endogenous transporter in which to hENT3 of cell the a of with of the transporter protein of and and forms of the respectively, previously of of with a intracellular with cell The of the the in from of the with The of with that or markers with the endosomal/lysosomal with hENT3 a of intracellular with with or markers and with the lysosomal the of the N-terminal dileucine motif in the of the a of the of proteins hENT3 hENT3 the first of the N-terminal region or hENT3 in which the dileucine motif and alanine contrast to the intracellular of in cell in or and intracellular in that with transcripts cell in with transcripts with of the N-terminal dileucine motif in the of of in proteins hENT3 hENT3 the first of the N-terminal region or hENT3 in which the dileucine motif and alanine to of in of with transcripts or or with and of hENT3 and of the of the ENT3 proteins the predominantly intracellular of hENT3 and its with the were in and the proteins and the intracellular targeting the of the and human and mouse proteins were that the of in were to in with the of mENT3, a hENT3 the of proteins, of were the intracellular targeting N-terminal of the protein or mutation of the of the motif to alanine and The the and identical the of the protein and the of the protein N-terminal The of the characterization of the of the the of in the of hENT3 a and contrast to of of the or the forms of in to in of and hENT3 in of in with the hENT3 transcripts or or in and or in and of of in with the transcripts or in and from to the of the transporter of and of which were the of the of and a of to of in The of the transporter of and and The in that hENT3 hENT1 and in a ENT2, the to ENT2, The of and the in and were and to and with respectively, of and The that and of of with transcripts or with and in of the of and of the of and and of in that in are of of in with the transcripts or in of and and of nucleoside and of a of nucleoside in The and were a of which from in its the in to a a of nucleoside the the that the and The and nucleoside in were the of a of with in the hENT1 the with a of and the and which of and with and of and that of were and with the of with of and of in with the transcripts or in were in in the or of the to of and the full-length of hENT3 and were respectively, from human and mouse of the that ENT3 widely in mouse and human tissues, and with a of the of ENT3 protein in mammalian of of region of identical to that of the protein and the are in The of in of the transporter protein with a particular abundance in and of and the to and forms of the respectively, were The of a from human and cell with to a of the hENT3 the of hENT3 transcripts in and The were found in a of tissues, and the were found in and of of from were and with The of proteins of are the of hENT3 and human and mouse family designated The 475-residue hENT3 and proteins were found to be 73% identical in amino acid sequence and and to the human and mouse a in the of the amino acid sequence of the ENT3 proteins are in the and that of the ENT2, and ENT3 in of the from the mammalian to of the from of the human and mouse ENT3 the of the family and are to the the mammalian with a and However, differ from the mammalian in a typical (DE)XXXL(LI) endosomal/lysosomal targeting motif in the hydrophilic region of the sequence that the first motif its in ENT3 from and dileucine motif found the in the ENT3 from the from the The of a lysosomal targeting motif that ENT3 intracellular a cell hENT3 were to the of the endogenous transporter in which to hENT3 of cell the a of with of the transporter protein of and and forms of the respectively, previously of of with a intracellular with cell The of the the in from of the with The of with that or markers with the endosomal/lysosomal with hENT3 a of intracellular with with or markers and with the lysosomal the of the N-terminal dileucine motif in the of the a of the of proteins hENT3 hENT3 the first of the N-terminal region or hENT3 in which the dileucine motif and alanine contrast to the intracellular of in cell in or and intracellular in that with transcripts cell in with transcripts with and of hENT3 and of the of the ENT3 proteins the predominantly intracellular of hENT3 and its with the were in and the proteins and the intracellular targeting the of the and human and mouse proteins were that the of in were to in with the of mENT3, a hENT3 the of proteins, of were the intracellular targeting N-terminal of the protein or mutation of the of the motif to alanine and The the and identical the of the protein and the of the protein N-terminal The of the characterization of the of the the of in the of hENT3 a and contrast to of of the or the forms of in to in the of the transporter of and of which were the of the of and a of to of in The of the transporter of and and The in that hENT3 hENT1 and in a ENT2, the to ENT2, The of and the in and were and to and with respectively, of and The that The of a of nucleoside in The and were a of which from in its the in to a a of nucleoside the the that the and The and nucleoside in were the of a of with in the hENT1 the with a of and the and which of and with and of and that of were and with the of with and the full-length of hENT3 and were respectively, from human and mouse of the that ENT3 widely in mouse and human tissues, and with a of the of ENT3 protein in mammalian of of region of identical to that of the protein and the are in The of in of the transporter protein with a particular abundance in and of and the to and forms of the respectively, were The of a from human and cell with to a of the hENT3 the of hENT3 transcripts in and The were found in a of tissues, and the were found in and and proteins are to be located the cell ENT1 in ENT1 and in the of cultured in the that the endogenous hENT3 proteins of cultured are predominantly located in intracellular with or of the transporter the cell of the of a of the human protein in in the intracellular of the protein and of cell targeting to the or the human and mouse ENT3 proteins, respectively, were in and found the lysosomal and hENT3 in the lysosomal and endogenous hENT3 hENT3 and the or that the transporter in part, in the of lysosomal previously a in membranes the of acid in the lysosomal to be the and in contrast to the equilibrative nucleoside transporter the ENT3 proteins of mouse, and a typical (DE)XXXL(LI) endosomal/lysosomal targeting motif The of motif in the intracellular targeting of ENT3 the of its to alanine or of the motif in its of the protein the human and mouse proteins, the of with transcripts the and the of of the dileucine motif alanine to in the of a of the transporter from the and cell to the plasma or mutation of the dileucine motif in which in of the transporter the a characterization to be of its and the transporter a from family members However, the to that of and to the of are to amino acid transporter of the of the the of the the the to the lysosomal of the of its of that the transporter and the respectively, and previously hENT1 the properties of nucleoside from are with the of of that in that to and the the plasma transporter hENT1 hENT1 and that the a of the of to of and and previously in the hENT1 to the of third that of the to be in nucleoside and characterized members of the family that to the plasma and be in the of from the lysosomal the transporters be to are with the nucleoside the lysosomal transporters. We to in the of the transporter to be or the of lysosomal transporters from of in lysosomal and acid transporter which and the of hENT3 to the and or of of in the transporters the which with nucleoside transporters and in the of and proteins are to be located the cell ENT1 in ENT1 and in the of cultured in the that the endogenous hENT3 proteins of cultured are predominantly located in intracellular with or of the transporter the cell of the of a of the human protein in in the intracellular of the protein and of cell targeting to the or the human and mouse ENT3 proteins, respectively, were in and found the lysosomal and hENT3 in the lysosomal and endogenous hENT3 hENT3 and the or that the transporter in part, in the of lysosomal previously a in membranes the of acid in the lysosomal to be the and in contrast to the equilibrative nucleoside transporter the ENT3 proteins of mouse, and a typical (DE)XXXL(LI) endosomal/lysosomal targeting motif The of motif in the intracellular targeting of ENT3 the of its to alanine or of the motif in its of the protein the human and mouse proteins, the of with transcripts the and the of of the dileucine motif alanine to in the of a of the transporter from the and cell to the plasma or mutation of the dileucine motif in which in of the transporter the a characterization to be of its and the transporter a from family members However, the to that of and to the of are to amino acid transporter of the of the the of the the the to the lysosomal of the of its of that the transporter and the respectively, and previously hENT1 the properties of nucleoside from are with the of of that in that to and the the plasma transporter hENT1 hENT1 and that the a of the of to of and and previously in the hENT1 to the of third that of the to be in nucleoside and characterized members of the family that to the plasma and be in the of from the lysosomal the transporters be to are with the nucleoside the lysosomal transporters. We to in the of the transporter to be or the of lysosomal transporters from of in lysosomal and acid transporter which and the of hENT3 to the and or of of in the transporters the which with nucleoside transporters and in the of We of and with with