Two Highly Conserved Glutamate Residues Critical for Type III Sodium-dependent Phosphate Transport Revealed by Uncoupling Transport Function from Retroviral Receptor Function

Abstract

Type III sodium-dependent phosphate (NaPi) cotransporters, Pit1 and Pit2, have been assigned housekeeping Pi transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaPicotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu55 and Glu575, with glutamine or with lysine severely impaired or knocked out, respectively, Pitransport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaPi cotransporters, in fungal NaPi cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaPicotransport. Their putative transmembranic positions together with the presented data are consistent with Glu55 and Glu575 being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 Pitransport function. Type III sodium-dependent phosphate (NaPi) cotransporters, Pit1 and Pit2, have been assigned housekeeping Pi transport functions and suggested involved in chondroblastic and osteoblastic mineralization and ectopic calcification. Both proteins exhibit dual function, thus, besides being transporters, they also serve as receptors for several gammaretroviruses. We here show that it is possible to uncouple transport and receptor functions of a type III NaPicotransporter and thus exploit the retroviral receptor function as a control for proper processing and folding of mutant proteins. Thus exchanging two putative transmembranic glutamate residues in human Pit2, Glu55 and Glu575, with glutamine or with lysine severely impaired or knocked out, respectively, Pitransport function, but left viral receptor function undisturbed. Both glutamates are conserved in type III NaPi cotransporters, in fungal NaPi cotransporters PHO-4 and Pho89, and in other known or putative phosphate permeases from a number of species and are the first residues shown to be critical for type III NaPicotransport. Their putative transmembranic positions together with the presented data are consistent with Glu55 and Glu575 being parts of a cation liganding site or playing roles in conformational changes associated with substrate transport. Finally, the results also show that Pit2 retroviral receptor function per se is not dependent on Pit2 Pitransport function. inorganic phosphate sodium-dependent phosphate murine leukemia virus amphotropic MLV an MLV isolate related to AM-MLV QQQ, etc., abbreviations for mutant Pit2 proteins where the three letters indicate the identity of the amino acids in positions 55, 91, and 575, respectively transmembrane Chinese hamster ovary phosphate-buffered saline N-hydroxysulfosuccinimide Inorganic phosphate (Pi)1 is essential for cellular metabolism and skeletal mineralization. Moreover, it serves as the source of phosphate for organic cell constituents, e.g. nucleotides and a variety of phosphorylated metabolic intermediates. Two proteins that show the same transport characteristics as Pi uptake across the plasma membrane in animal cells have been identified (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 2Olah Z. Lehel C. Anderson W.B. Eiden M.V. Wilson C.A. J. Biol. Chem. 1994; 269: 25426-25431Abstract Full Text PDF PubMed Google Scholar, 3Wilson C.A. Eiden M.V. Anderson W.B. Lehel C. Olah Z. J. Virol. 1995; 69: 534-537Crossref PubMed Google Scholar), namely the sodium-dependent phosphate (NaPi) cotransporters, Pit1 (human Pit1 formerly GLVR1 (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar)) and Pit2 (human Pit2 formerly GLVR2 (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar)). Both proteins are characterized as type III NaPi cotransporters (6Kavanaugh M.P. Kabat D. Kidney Int. 1996; 49: 959-963Abstract Full Text PDF PubMed Scopus (159) Google Scholar) and show a broad tissue distribution being expressed in all investigated human tissues albeit at different levels (7Uckert W. Willimsky G. Pedersen F.S. Blankenstein T. Pedersen L. Hum. Gene Ther. 1998; 9: 2619-2627Crossref PubMed Google Scholar). Furthermore, low extracellular Pilevels result in up-regulated Pit1 and Pit2 expression in mammalian cells (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 8Chien M.L. O'Neill E. Garcia J.V. Virology. 1998; 240: 109-117Crossref PubMed Scopus (26) Google Scholar). These observations that the cellular Pi in mammalian cells is type III NaPi cotransporters (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar). results also at type III as playing roles in chondroblastic and osteoblastic mineralization Pedersen F.S. Pedersen L. PubMed Scopus Google Scholar, G. J. J. W. J. PubMed Scopus Google Scholar) as as being involved in are in and with S. A. H. PubMed Google Scholar). the roles of type III NaPi are not that extracellular Pi levels expression of the for and that the is dependent on Pi uptake across the plasma membrane B. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is involved in and in J. Full Text Full Text PDF PubMed Scopus Google the roles of type III NaPi cotransporters in cellular Pi uptake and for critical roles in and is known transport function. two known type III NaPi cotransporters, Pit1 and Pit2, show amino identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar), of Pit1 and Pit2 exhibit amino identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, S.V. O'Hara B. J. Virol. PubMed Google Scholar, C.A. Eiden M.V. J. Virol. 1994; PubMed Google Scholar). putative for proteins on transmembrane and extracellular (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, S.V. O'Hara B. J. Virol. PubMed Google Scholar, M.L. Garcia J.V. J. Virol. PubMed Google Scholar). type III cotransporters and of the two other NaPi to as and (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Pit1 and Pit2 identified as receptors for to the and they are the two cellular receptors in human retroviral human Pit1 as a receptor for leukemia virus (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar) and leukemia virus G. O'Hara B. J. Virol. PubMed Google Scholar), and the from human and as receptor for amphotropic murine leukemia virus (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, D.G. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google an as receptor D.G. Miller A.D. J. Virol. 1994; PubMed Google Scholar). the receptor functions of Pit1 and Pit2 for been in J. Miller A.D. Eiden M.V. Biol. PubMed Scopus Google and J. Virol. PubMed Scopus Google that the retroviral receptor function a for proper processing and folding of mutant type III NaPi with knocked function thus for of amino acids critical for transport function. We here show that it is possible to uncouple human Pit2 retroviral receptor function from function and to exploit the dual function of Pit2 as NaPi and retroviral receptor for of amino acids critical for NaPi transport. identified two Pit2 glutamate residues Glu55 and Glu575 as critical for Pi transport function. glutamates are in putative transmembranic and are conserved in type III NaPi cotransporters, in NaPi cotransporters PHO-4 from Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) and from P. 1998; PubMed Scopus Google Scholar), and in a number of known and putative phosphate permeases from other species that they also are critical for the function of III NaPi cotransporters, Pit1 and Pit2, exhibit dual function. have shown that it is possible to Pi transport function of human Pit2, retroviral receptor function undisturbed. it is possible to exploit the retroviral receptor function of Pit2, and of as a control for processing and folding of mutant proteins with knocked transport function. identified two glutamate and in human Pit2, as critical for Pit2 Pitransport function. is the first amino acids critical for Pi transport of a type III NaPi have been two glutamates are conserved in type III NaPi cotransporters and in a number of known and putative phosphate permeases from other species that they also are critical for the function of of retroviral receptor function as a control for processing and folding of mutant Pit2 proteins on expression in is with the mutant severely impaired transport function, not be the not being in the but it also show impaired receptor function. is possible that impaired receptor function be to in retroviral of the amino acids in it is not mutant proteins and that the mutant proteins in the plasma membrane exhibit processing viral receptor function be as control of extracellular amino acids in transport function of type III is in that the amino acids identified as critical for retroviral receptor function of Pit1 and Pit2 are Pit1 and Pit2 and the NaPi PHO-4 from D.G. Miller A.D. J. Virol. 1994; PubMed Google Scholar, L. Johann S.V. Zeijl M. Pedersen F.S. O'Hara B. J. Virol. 1995; 69: PubMed Google Scholar, Pedersen F.S. Pedersen L. J. Virol. PubMed Scopus Google Scholar, M.V. Wilson C.A. J. Virol. 1996; PubMed Google Scholar, Eiden M.V. Wilson C.A. J. Virol. 1998; PubMed Google Scholar, Pedersen F.S. O'Hara B. Pedersen L. J. Virol. PubMed Google Scholar, L. Zeijl M. Johann S.V. O'Hara B. J. Virol. PubMed Google Scholar, A. Kabat D. J. Virol. PubMed Scopus Google Scholar), the amino acids critical for NaPi uptake are to be the residues conserved in Pit2, and related proteins from other species as shown in the with have shown that a Pit1 mutant with receptor functions in Pi transport function as D. P. and L. it is possible that in is critical for Pi transport function and critical for receptor not the of Pit2 residues and in Pi transport. the that or for lysine to Pitransport function receptor function is in with residues being involved in function, is possible that and are involved in conformational changes of the data also consistent with glutamates being parts of a cation liganding site residues from different transmembrane as been for residues of the the of the and P. L. 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. J. C. G. T. 1996; PubMed Scopus Google Scholar, B. 1995; PubMed Scopus Google Scholar, G. U. P. PubMed Scopus Google Scholar). a site the other residues e.g. glutamine and G. U. P. PubMed Scopus Google Scholar). it is to that at of the mutant Pit2 proteins with for in of the positions or and low levels of NaPi uptake a number of transport proteins have been identified that have dual function as and retroviral receptors (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar, Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google D.G. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, L. D. Cunningham Full Text PDF PubMed Scopus Google Scholar, Miller A.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. Kabat D. J. Virol. PubMed Google Scholar, Kabat D. J. Virol. PubMed Google Scholar). murine is a amino it also been shown that a glutamate in a putative transmembrane critical for the transport function but not for retroviral receptor function of the H. M.P. Kabat D. Virology. 1994; PubMed Scopus (26) Google Scholar). on the data presented here and that of H. M.P. Kabat D. Virology. 1994; PubMed Scopus (26) Google Scholar), that for proteins dual function as and retroviral viral receptor function in be for of amino residues critical for transport function. a is a and for the viral receptor function as it a to of mutant proteins receptor presented data also have for the of Pit2 in retroviral of to cells shown to Pit2 Pi transport and AM-MLV but not virus P. J. Virol. PubMed Google Scholar). to the data that conformational changes in Pit2 are involved in AM-MLV Pit2 P. J. Virol. PubMed Google Scholar). data are not in with but they show that viral receptor function is not dependent on Pi transport of the data in and that is Pi transport function and retroviral receptor function of We that functions of the type III be on transport and receptor functions as presented Inorganic phosphate (Pi)1 is essential for cellular metabolism and skeletal mineralization. Moreover, it serves as the source of phosphate for organic cell constituents, e.g. nucleotides and a variety of phosphorylated metabolic intermediates. Two proteins that show the same transport characteristics as Pi uptake across the plasma membrane in animal cells have been identified (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 2Olah Z. Lehel C. Anderson W.B. Eiden M.V. Wilson C.A. J. Biol. Chem. 1994; 269: 25426-25431Abstract Full Text PDF PubMed Google Scholar, 3Wilson C.A. Eiden M.V. Anderson W.B. Lehel C. Olah Z. J. Virol. 1995; 69: 534-537Crossref PubMed Google Scholar), namely the sodium-dependent phosphate (NaPi) cotransporters, Pit1 (human Pit1 formerly GLVR1 (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar)) and Pit2 (human Pit2 formerly GLVR2 (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar)). Both proteins are characterized as type III NaPi cotransporters (6Kavanaugh M.P. Kabat D. Kidney Int. 1996; 49: 959-963Abstract Full Text PDF PubMed Scopus (159) Google Scholar) and show a broad tissue distribution being expressed in all investigated human tissues albeit at different levels (7Uckert W. Willimsky G. Pedersen F.S. Blankenstein T. Pedersen L. Hum. Gene Ther. 1998; 9: 2619-2627Crossref PubMed Google Scholar). Furthermore, low extracellular Pilevels result in up-regulated Pit1 and Pit2 expression in mammalian cells (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar, 8Chien M.L. O'Neill E. Garcia J.V. Virology. 1998; 240: 109-117Crossref PubMed Scopus (26) Google Scholar). These observations that the cellular Pi in mammalian cells is type III NaPi cotransporters (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar). results also at type III as playing roles in chondroblastic and osteoblastic mineralization Pedersen F.S. Pedersen L. PubMed Scopus Google Scholar, G. J. J. W. J. PubMed Scopus Google Scholar) as as being involved in are in and with S. A. H. PubMed Google Scholar). the roles of type III NaPi are not that extracellular Pi levels expression of the for and that the is dependent on Pi uptake across the plasma membrane B. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is involved in and in J. Full Text Full Text PDF PubMed Scopus Google Scholar). the roles of type III NaPi cotransporters in cellular Pi uptake and for critical roles in and is known transport function. two known type III NaPi cotransporters, Pit1 and Pit2, show amino identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar), of Pit1 and Pit2 exhibit amino identity (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, S.V. O'Hara B. J. Virol. PubMed Google Scholar, C.A. Eiden M.V. J. Virol. 1994; PubMed Google Scholar). putative for proteins on transmembrane and extracellular (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, S.V. O'Hara B. J. Virol. PubMed Google Scholar, M.L. Garcia J.V. J. Virol. PubMed Google Scholar). type III cotransporters and of the two other NaPi to as and (1Kavanaugh M.P. Miller D.G. Zhang W. Law W. Kozak S.L. Kabat D. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7071-7075Crossref PubMed Scopus (526) Google Scholar). Both Pit1 and Pit2 identified as receptors for to the and they are the two cellular receptors in human retroviral human Pit1 as a receptor for leukemia virus (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar) and leukemia virus G. O'Hara B. J. Virol. PubMed Google Scholar), and the from human and as receptor for amphotropic murine leukemia virus (5van Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google Scholar, D.G. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google an as receptor D.G. Miller A.D. J. Virol. 1994; PubMed Google Scholar). the receptor functions of Pit1 and Pit2 for been in J. Miller A.D. Eiden M.V. Biol. PubMed Scopus Google and J. Virol. PubMed Scopus Google Scholar). We that the retroviral receptor function a for proper processing and folding of mutant type III NaPi with knocked function thus for of amino acids critical for transport function. We here show that it is possible to uncouple human Pit2 retroviral receptor function from function and to exploit the dual function of Pit2 as NaPi and retroviral receptor for of amino acids critical for NaPi transport. identified two Pit2 glutamate residues Glu55 and Glu575 as critical for Pi transport function. glutamates are in putative transmembranic and are conserved in type III NaPi cotransporters, in NaPi cotransporters PHO-4 from Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) and from P. 1998; PubMed Scopus Google Scholar), and in a number of known and putative phosphate permeases from other species that they also are critical for the function of III NaPi cotransporters, Pit1 and Pit2, exhibit dual function. have shown that it is possible to Pi transport function of human Pit2, retroviral receptor function undisturbed. it is possible to exploit the retroviral receptor function of Pit2, and of as a control for processing and folding of mutant proteins with knocked transport function. identified two glutamate and in human Pit2, as critical for Pit2 Pitransport function. is the first amino acids critical for Pi transport of a type III NaPi have been two glutamates are conserved in type III NaPi cotransporters and in a number of known and putative phosphate permeases from other species that they also are critical for the function of of retroviral receptor function as a control for processing and folding of mutant Pit2 proteins on expression in is with the mutant severely impaired transport function, not be the not being in the but it also show impaired receptor function. is possible that impaired receptor function be to in retroviral of the amino acids in it is not mutant proteins and that the mutant proteins in the plasma membrane exhibit processing viral receptor function be as control of extracellular amino acids in transport function of type III is in that the amino acids identified as critical for retroviral receptor function of Pit1 and Pit2 are Pit1 and Pit2 and the NaPi PHO-4 from D.G. Miller A.D. J. Virol. 1994; PubMed Google Scholar, L. Johann S.V. Zeijl M. Pedersen F.S. O'Hara B. J. Virol. 1995; 69: PubMed Google Scholar, Pedersen F.S. Pedersen L. J. Virol. PubMed Scopus Google Scholar, M.V. Wilson C.A. J. Virol. 1996; PubMed Google Scholar, Eiden M.V. Wilson C.A. J. Virol. 1998; PubMed Google Scholar, Pedersen F.S. O'Hara B. Pedersen L. J. Virol. PubMed Google Scholar, L. Zeijl M. Johann S.V. O'Hara B. J. Virol. PubMed Google Scholar, A. Kabat D. J. Virol. PubMed Scopus Google Scholar), the amino acids critical for NaPi uptake are to be the residues conserved in Pit2, and related proteins from other species as shown in the with have shown that a Pit1 mutant with receptor functions in Pi transport function as D. P. and L. it is possible that in is critical for Pi transport function and critical for receptor not the of Pit2 residues and in Pi transport. the that or for lysine to Pitransport function receptor function is in with residues being involved in function, is possible that and are involved in conformational changes of the data also consistent with glutamates being parts of a cation liganding site residues from different transmembrane as been for residues of the the of the and P. L. 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. J. C. G. T. 1996; PubMed Scopus Google Scholar, B. 1995; PubMed Scopus Google Scholar, G. U. P. PubMed Scopus Google Scholar). a site the other residues e.g. glutamine and G. U. P. PubMed Scopus Google Scholar). it is to that at of the mutant Pit2 proteins with for in of the positions or and low levels of NaPi uptake a number of transport proteins have been identified that have dual function as and retroviral receptors (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar, Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google D.G. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, L. D. Cunningham Full Text PDF PubMed Scopus Google Scholar, Miller A.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. Kabat D. J. Virol. PubMed Google Scholar, Kabat D. J. Virol. PubMed Google Scholar). murine is a amino it also been shown that a glutamate in a putative transmembrane critical for the transport function but not for retroviral receptor function of the H. M.P. Kabat D. Virology. 1994; PubMed Scopus (26) Google Scholar). on the data presented here and that of H. M.P. Kabat D. Virology. 1994; PubMed Scopus (26) Google Scholar), that for proteins dual function as and retroviral viral receptor function in be for of amino residues critical for transport function. a is a and for the viral receptor function as it a to of mutant proteins receptor presented data also have for the of Pit2 in retroviral of to cells shown to Pit2 Pi transport and AM-MLV but not virus P. J. Virol. PubMed Google Scholar). to the data that conformational changes in Pit2 are involved in AM-MLV Pit2 P. J. Virol. PubMed Google Scholar). data are not in with but they show that viral receptor function is not dependent on Pi transport of the data in and that is Pi transport function and retroviral receptor function of We that functions of the type III be on transport and receptor functions as presented Type III NaPi cotransporters, Pit1 and Pit2, exhibit dual function. have shown that it is possible to Pi transport function of human Pit2, retroviral receptor function undisturbed. it is possible to exploit the retroviral receptor function of Pit2, and of as a control for processing and folding of mutant proteins with knocked transport function. identified two glutamate and in human Pit2, as critical for Pit2 Pitransport function. is the first amino acids critical for Pi transport of a type III NaPi have been two glutamates are conserved in type III NaPi cotransporters and in a number of known and putative phosphate permeases from other species that they also are critical for the function of of retroviral receptor function as a control for processing and folding of mutant Pit2 proteins on expression in is with the mutant severely impaired transport function, not be the not being in the but it also show impaired receptor function. is possible that impaired receptor function be to in retroviral of the amino acids in it is not mutant proteins and that the mutant proteins in the plasma membrane exhibit processing viral receptor function be as control of extracellular amino acids in transport function of type III is in that the amino acids identified as critical for retroviral receptor function of Pit1 and Pit2 are Pit1 and Pit2 and the NaPi PHO-4 from D.G. Miller A.D. J. Virol. 1994; PubMed Google Scholar, L. Johann S.V. Zeijl M. Pedersen F.S. O'Hara B. J. Virol. 1995; 69: PubMed Google Scholar, Pedersen F.S. Pedersen L. J. Virol. PubMed Scopus Google Scholar, M.V. Wilson C.A. J. Virol. 1996; PubMed Google Scholar, Eiden M.V. Wilson C.A. J. Virol. 1998; PubMed Google Scholar, Pedersen F.S. O'Hara B. Pedersen L. J. Virol. PubMed Google Scholar, L. Zeijl M. Johann S.V. O'Hara B. J. Virol. PubMed Google Scholar, A. Kabat D. J. Virol. PubMed Scopus Google Scholar), the amino acids critical for NaPi uptake are to be the residues conserved in Pit2, and related proteins from other species as shown in the with have shown that a Pit1 mutant with receptor functions in Pi transport function as D. P. and L. it is possible that in is critical for Pi transport function and critical for receptor function. We not the of Pit2 residues and in Pi transport. the that or for lysine to Pitransport function receptor function is in with residues being involved in function, is possible that and are involved in conformational changes of the data also consistent with glutamates being parts of a cation liganding site residues from different transmembrane as been for residues of the the of the and P. L. 1998; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, B. J. C. G. T. 1996; PubMed Scopus Google Scholar, B. 1995; PubMed Scopus Google Scholar, G. U. P. PubMed Scopus Google Scholar). a site the other residues e.g. glutamine and G. U. P. PubMed Scopus Google Scholar). it is to that at of the mutant Pit2 proteins with for in of the positions or and low levels of NaPi uptake a number of transport proteins have been identified that have dual function as and retroviral receptors (4O'Hara B. Johann S.V. Klinger H.P. Blair D.G. Rubinson H. Dunn K.J. Sass P. Vitek S.M. Robins T. Cell Growth Differ. 1990; 1: 119-127PubMed Google Scholar, Zeijl M. Johann S.V. Closs E. Cunningham J. Eddy R. Shows T.B. O'Hara B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1168-1172Crossref PubMed Scopus (240) Google D.G. Miller A.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, L. D. Cunningham Full Text PDF PubMed Scopus Google Scholar, Miller A.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. Kabat D. J. Virol. PubMed Google Scholar, Kabat D. J. Virol. PubMed Google Scholar). murine is a amino it also been shown that a glutamate in a putative transmembrane critical for the transport function but not for retroviral receptor function of the H. M.P. Kabat D. Virology. 1994; PubMed Scopus (26) Google Scholar). on the data presented here and that of H. M.P. Kabat D. Virology. 1994; PubMed Scopus (26) Google Scholar), that for proteins dual function as and retroviral viral receptor function in be for of amino residues critical for transport function. a is a and for the viral receptor function as it a to of mutant proteins receptor function. presented data also have for the of Pit2 in retroviral of to cells shown to Pit2 Pi transport and AM-MLV but not virus P. J. Virol. PubMed Google Scholar). to the data that conformational changes in Pit2 are involved in AM-MLV Pit2 P. J. Virol. PubMed Google Scholar). data are not in with but they show that viral receptor function is not dependent on Pi transport of the data in and that is Pi transport function and retroviral receptor function of We that functions of the type III be on transport and receptor functions as presented We O'Hara for Eiden for the cell Garcia for human and for of We for and with with