Glycosylation Is Important for Cell Surface Expression of the Water Channel Aquaporin-2 but Is Not Essential for Tetramerization in the Endoplasmic Reticulum

Aquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that ∼25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wild-type AQP2 are stable proteins with a half-life of 6-12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus. Aquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that ∼25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wild-type AQP2 are stable proteins with a half-life of 6-12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus. Aquaporins (AQPs) 1The abbreviations used are: AQP(s), aquaporin(s); BFA, brefeldin A; CHO cells, Chinese hamster ovary cells; EGFP, enhanced green fluorescent protein; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GFP, green fluorescent protein; MDCK cells, Madin-Darby canine kidney cells; NDI, nephrogenic diabetes insipidus; PNGase F, peptide N-glycosidase F; SNARE, soluble NSF attachment protein receptors; TGN, trans-Golgi network; PDI, protein disulfide isomerase.1The abbreviations used are: AQP(s), aquaporin(s); BFA, brefeldin A; CHO cells, Chinese hamster ovary cells; EGFP, enhanced green fluorescent protein; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GFP, green fluorescent protein; MDCK cells, Madin-Darby canine kidney cells; NDI, nephrogenic diabetes insipidus; PNGase F, peptide N-glycosidase F; SNARE, soluble NSF attachment protein receptors; TGN, trans-Golgi network; PDI, protein disulfide isomerase. are ubiquitously expressed pore-forming proteins that facilitate water transport across membranes along an osmotic gradient (see Ref. 1.Kozono D. Yasui M. King L.S. Agre P. J. Clin. Invest. 2002; 109: 1395-1399Crossref PubMed Scopus (216) Google Scholar). AQPs share a highly conserved domain organization with six transmembrane domains and cytoplasmically oriented N and C termini. AQPs form tetramers or sometimes higher order oligomers in membranes. Structural studies of AQP1 revealed that the functional unit is a tetramer with each monomer providing an independent water pore (2.Murata K. Mitsuoka K. Hirai T. Walz T. Agre P. Heymann J.B. Engel A. Fujiyoshi Y. Nature. 2000; 407: 599-605Crossref PubMed Scopus (1394) Google Scholar). Stability of the tetramer is thought to be conferred by the assembly of monomers as a tight fitting wedge within the tetramer. With the exception of AQP2, which is regulated by the antidiuretic hormone vasopressin, most of the AQPs are expressed constitutively at the plasma membrane (see Ref. 3.Nielsen S. Frokjaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1005) Google Scholar). AQP2 is localized in a tubulovesicular subapical storage compartment. The intracellular retention is thought to be caused by tethering of the storage vesicles to the actin cytoskeleton and is regulated by members of the rho subfamily of small GTPases (4.Klussmann E. Tamma G. Lorenz D. Wiesner B. Maric K. Hofmann F. Aktories K. Valenti G. Rosenthal W. J. Biol. Chem. 2001; 276: 20451-20457Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Binding of vasopressin to the basolateral V2 receptor increases cAMP concentration which activates protein kinase A. Protein kinase A phosphorylates AQP2-Ser256 which is required for fusion of AQP2 storage vesicles with the plasma membrane (5.Fushimi K. Sasaki S. Marumo F. J. Biol. Chem. 1997; 272: 14800-14804Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 6.van Balkom B.W.M. Savelkoul P.J.M. Markovich D. Hofman E. Nielsen S. van der Sluijs P. Deen P.M.T. J. Biol. Chem. 2002; 277: 41473-41479Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The precise mechanism responsible for the recruitment of tetrameric AQP2 to the cell surface is not known, but phosphorylation of at least three of the four monomers, reportedly, is required and sufficient for the localization of AQP2 on the plasma membrane (7.Kamsteeg E.J. Heijnen I. van Os C.H. Deen P.M.T. J. Cell Biol. 2000; 151: 919-929Crossref PubMed Scopus (147) Google Scholar). The regulated recruitment of plasma membrane proteins from intracellular storage vesicles provides an efficient means to control cell surface expression and was originally discovered for the insulin-regulated glucose transporter GLUT4 (see Ref. 8.Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (907) Google Scholar). GLUT4 and AQP2 translocation share some similarities such as the requirement for the v-SNARE synaptobrevin-2 in fusion with the plasma membrane (8.Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (907) Google Scholar, 9.Gouraud S. Laera A. Calmita G. Carmosini M. Procino G. Rosetto O. Mannucci R. Rosenthal W. Svelto M. Valenti G. J. Cell Sci. 2002; 115: 3667-3674Crossref PubMed Scopus (63) Google Scholar). Nevertheless, there are important differences. For instance, GLUT4 translocates from a basolateral storage compartment in polarized epithelial cells, and phosphorylation of GLUT4 inhibits glucose transport, whereas phosphorylation of AQP2 is essential for its recruitment from an apical storage compartment. Despite the progress that has been made in understanding the physiology of AQP-mediated water transport, important aspects such as biosynthetic maturation and intracellular transport of AQP water channels are incompletely understood. For instance, it has been shown for AQP1 that conformational changes and rearrangements take place during insertion in the endoplasmic reticulum (ER), but whether insertion occurs cotranslationally or post-translationally has become a controversial issue (10.Skach W.R. Shi L. Calayag M.C. Frigeri A. Lingapppa V.R. Verkman A.S. J. Cell Biol. 1994; 125: 803-815Crossref PubMed Scopus (68) Google Scholar, 11.Dohke Y. Turner R.J. J. Biol. Chem. 2002; 277: 15215-15219Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In addition, with the exception of AQP4 (12.Madrid R. Le Maout S. Barrault M.B. Janvier K. Benichou S. J. J. 2001; PubMed Scopus Google the sorting required for transport of AQPs intracellular to be defined. The that AQP2 and M. 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Full Text PDF PubMed Scopus Google stable CHO AQP2 or to shown in was localized in an intracellular compartment and was not on the plasma whereas wild-type AQP2 both on the cell surface and as in MDCK cells. of for a small on the localization of wild-type AQP2, which with the membrane in a for that it caused of the and a of the Golgi protein as was shown by Full Text PDF PubMed Scopus Google Scholar, J. E. M. G. Nature. 2000; 407: PubMed Scopus Google Scholar). localization was by from the in the control to a in the which also results to h with is of to that the in control with the of Golgi with with wild-type be with that a of intracellular wild-type AQP2 is in storage vesicles a compartment. the and show that glycosylation of AQP2 is not essential for from the but is a for exit from the Golgi complex. of AQP2 in the and AQP2 forms a functional water pore in the apical plasma membrane. of transport channels has been to both in the K. A. P. S. S. D. P. J. Cell Biol. 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Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). are to the molecular mechanisms and transport directing of wild-type AQP2 from the to the Golgi complex and have not been defined. that of newly synthesized AQP2 is glycosylated. in the glycosylation was found for AQP1 Agre P. J. Clin. Invest. 1994; PubMed Google whereas of AQP2 was R. van van Os C.H. Deen P.M.T. J. PubMed Google Scholar). The glycosylation of AQP2 is caused by that are by the of the glycosylation to the transmembrane with that the of glycosylation of such increases with the in the G. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). for is by the results of the with wild-type AQP2, of is at the of the the h of the the of the that is that the of the in the with that of wild-type AQP2 with the for A of AQP2 that are in the that have N-linked glycosylation S. Savelkoul P.J.M. F. M. D. A. Rosenthal W. D. van Os C.H. Deen P.M.T. 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P. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the half-life of newly synthesized AQP2 is in the order of h ∼25% of the AQP2 is at the of a is a as found that the synthesis of AQP2 in MDCK is in on These AQP2 are beyond the ER, are not to and most in the subapical storage The of vasopressin or is the of a cAMP which results in the phosphorylation of in AQP2 and enhanced of storage vesicles to the apical cell surface. wild-type AQP2 in the storage be it or is a for protein kinase A and be to the cell surface. In the that is not beyond the Golgi complex reaches storage compartment and to be to the cell surface in the of cAMP glycosylation is important for protein glycosylation is the most is the of proteins that not a functional (see Ref. L. A. Nat. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). not for on N-linked that have a on protein on the of the N-linked glycosylation of the glycosylation in a protein with a it is of to that the was found for and in the glycosylation is from wild-type AQP2 glycosylation is not important for AQP2 that AQP2 is not on the control mechanisms L. A. Nat. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). control mechanisms have been for the and sorting of proteins to In the transmembrane has been which and membrane proteins with transmembrane domains to for F. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). are not in transmembrane are in membrane AQP2 is not to be the of found that the intracellular of water in MDCK is on the of N-linked In the of AQP2 to the Golgi complex but not the apical plasma membrane. glycosylation and are thought to be in apical as was shown for proteins P. J. K. Nature. PubMed Scopus Google Scholar, A. F. K. J. PubMed Scopus Google Scholar). and B. Verkman A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google found that AQP2 is soluble in at not to a in apical transport of is that the of AQP2 an essential in its sorting in the Golgi complex. of to the and which are localized in has been to be important for sorting (see Ref. M. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). the of to be by localized to the O. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). or in addition, of AQP2 from the and its apical transport vesicles of its with a (see Ref. K. K. Full Text PDF PubMed Scopus Google Scholar). control there be the and wild-type AQP2 that to the transport of the that in of the precise it is that glycosylation of AQP2 is important for its to as a water on the cell surface. The be a mechanism with the to the plasma membrane. are to We are to and for the and for We and for providing