Domain Interactions of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor
2005; Elsevier BV; Volume: 280; Issue: 22 Linguagem: Inglês
10.1074/jbc.m412971200
ISSN1083-351X
AutoresJodi L. Kreiling, James C. Byrd, Richard G. MacDonald,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. α-FLAG or α-Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the α-FLAG but not α-Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the α-FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor. The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. α-FLAG or α-Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the α-FLAG but not α-Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the α-FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor. The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) 1The abbreviations used are: M6P/IGF2R, mannose 6-phosphate/insulin-like growth factor II receptor; Man-6-P, mannose 6-phosphate; EC, extracytoplasmic; TGF-β1, transforming growth factor-β1; IGF-II, insulin-like growth factor II; uPAR, urokinase-type plasminogen activator receptor; α-, anti-; PMP-BSA, pentamannose phosphate-bovine serum albumin; hGUS, human β-glucuronidase; WT-M, full-length wild-type M6P/IGF2R-Myc; R2AxI/T-M, full-length triple-mutant M6P/IGF2R-Myc; nt, nucleotide(s); pBSKII+, pBluescript SK II+; DTT, dithiothreitol; HBS, HEPES-buffered saline; EGFR, epidermal growth factor receptor.1The abbreviations used are: M6P/IGF2R, mannose 6-phosphate/insulin-like growth factor II receptor; Man-6-P, mannose 6-phosphate; EC, extracytoplasmic; TGF-β1, transforming growth factor-β1; IGF-II, insulin-like growth factor II; uPAR, urokinase-type plasminogen activator receptor; α-, anti-; PMP-BSA, pentamannose phosphate-bovine serum albumin; hGUS, human β-glucuronidase; WT-M, full-length wild-type M6P/IGF2R-Myc; R2AxI/T-M, full-length triple-mutant M6P/IGF2R-Myc; nt, nucleotide(s); pBSKII+, pBluescript SK II+; DTT, dithiothreitol; HBS, HEPES-buffered saline; EGFR, epidermal growth factor receptor. is a multifunctional member of the p-lectin family and is a type 1 integral membrane glycoprotein of ∼300 kDa (1Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (770) Google Scholar, 2Hassan A.B. Am. J. Pathol. 2003; 162: 3-6Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This receptor comprises a large extracytoplasmic (EC) domain, a single membrane-spanning region, and a short cytoplasmic tail. The EC domain is the principal ligand-binding region of the receptor, consisting of 15 homologous repeats of ∼145 amino acids each (1Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (770) Google Scholar, 3Oshima A. Nolan C.M. Kyle J.W. Grubb J.H. Sly W.S. J. Biol. Chem. 1988; 263: 2553-2562Abstract Full Text PDF PubMed Google Scholar, 4Lobel P. Dahms N.M. Kornfeld S. J. Biol. Chem. 1988; 263: 2563-2570Abstract Full Text PDF PubMed Google Scholar). The M6P/IGF2R has been shown to bind at least two classes of ligands, the Man-6-P-containing and the non-Man-6-P-containing polypeptide ligands, all of which bind to sites within the EC region (5Dahms N.M. Lobel P. Kornfeld S. J. Biol. Chem. 1989; 264: 12115-12118Abstract Full Text PDF PubMed Google Scholar, 6Dahms N.M. Biochem. Soc. Trans. 1996; 24: 136-141Crossref PubMed Scopus (38) Google Scholar, 7Braulke T. Horm. Metab. Res. 1999; 31: 242-246Crossref PubMed Scopus (132) Google Scholar). Newly synthesized Man-6-P-containing ligands such as lysosomal acid hydrolases bind to the M6P/IGF2R in the trans-Golgi network through Man-6-P residues on their N-linked oligosaccharides, whereas other Man-6-P-containing ligands, such as latent transforming growth factor-β1 (TGF-β1), proliferin, and granzyme B, bind at the cell surface (1Ghosh P. Dahms N.M. Kornfeld S. Nat. Rev. Mol. Cell Biol. 2003; 4: 202-212Crossref PubMed Scopus (770) Google Scholar, 8Dahms N.M. Hancock M.K. Biochim. Biophys. Acta. 2002; 1572: 317-340Crossref PubMed Scopus (179) Google Scholar). Through binding of this large class of ligands, the M6P/IGF2R mediates several important cellular functions, such as the endocytosis and/or targeting of acid hydrolases to lysosomes (9Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (923) Google Scholar), the proteolytic activation of latent TGF-β1 (10Purchio A.F. Cooper J.A. Brunner A.M. Lioubin M.N. Gentry L.E. Kovacina K.S. Roth R.A. Marquardt H. J. Biol. Chem. 1988; 263: 14211-14215Abstract Full Text PDF PubMed Google Scholar, 11Dennis P.A. Rifkin D.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 580-584Crossref PubMed Scopus (457) Google Scholar, 12Ghahary A. Tredget E.E. Shen Q. J. Cell. Physiol. 1999; 180: 61-70Crossref PubMed Scopus (29) Google Scholar), mediation of the migration and angiogenesis induced by proliferin (13Groskopf J.C. Syu L.J. Saltiel A.R. Linzer D.I. Endocrinology. 1997; 138: 2835-2840Crossref PubMed Scopus (65) Google Scholar), and the internalization of granzyme B (14Motyka B. Korbutt G. Pinkoski M.J. Heibein J.A. Caputo A. Hobman M. Barry M. Shostak I. Sawchuk T. Holmes C.F. Gauldie J. Bleackley R.C. Cell. 2000; 103: 491-500Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Two distinct high affinity binding sites and one, recently discovered, low affinity binding site for the Man-6-P-containing ligands map to specific residues that are common to repeats 3 and 9 and repeat 5 of the EC domain, respectively (15Westlund B. Dahms N.M. Kornfeld S. J. Biol. Chem. 1991; 266: 23233-23239Abstract Full Text PDF PubMed Google Scholar, 16Dahms N.M. Rose P.A. Molkentin J.D. Zhang Y. Brzycki M.A. J. Biol. Chem. 1993; 268: 5457-5463Abstract Full Text PDF PubMed Google Scholar, 17Olson L.J. Zhang J. Lee Y.C. Dahms N.M. Kim J.J. J. Biol. Chem. 1999; 274: 29889-29896Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 18Hancock M.K. Haskins D.J. Sun G. Dahms N.M. J. Biol. Chem. 2002; 277: 11255-11264Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 19Reddy S.T. Chai W. Childs R.A. Page J.D. Feizi T. Dahms N.M. J. Biol. Chem. 2004; 279: 38658-38667Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The two high affinity binding sites are not functionally equivalent with respect to ligand preference, having distinct dissociation constants for the multivalent Man-6-P-ligand β-glucuronidase (2.0 versus 4.3 nm for repeats 3 and 9, respectively) (20Marron-Terada P.G. Brzycki-Wessell M.A. Dahms N.M. J. Biol. Chem. 1998; 273: 22358-22366Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The pH optimum for carbohydrate binding is also more acidic for repeat 9 than repeat 3 (pH 6.4 versus pH 6.9, respectively), and the two sites differ in their ability to recognize distinctive modifications found on Dictyostelium discoideum glycoproteins, such as mannose 6-sulfate and Man-6-P methyl esters (21Marron-Terada P.G. Hancock M.K. Haskins D.J. Dahms N.M. Biochemistry. 2000; 39: 2243-2253Crossref PubMed Scopus (36) Google Scholar). Additionally, repeat 9 alone can fold into a high affinity ligand binding domain, whereas repeat 3 depends on residues in adjacent repeats 1 and/or 2 for optimal ligand binding (22Hancock M.K. Yammani R.D. Dahms N.M. J. Biol. Chem. 2002; 277: 47205-47212Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Although it exhibits significant sequence homology with repeats 3 and 9, as well as sharing four conserved residues key for Man-6-P binding, repeat 5 has an ∼300-fold lower affinity for Man-6-P than repeat 9 or repeats 1–3, possibly due to the absence of two half-cystines that form a stabilizing disulfide bond in repeats 3 and 9 (19Reddy S.T. Chai W. Childs R.A. Page J.D. Feizi T. Dahms N.M. J. Biol. Chem. 2004; 279: 38658-38667Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).The non-Man-6-P-containing class of ligands includes the polypeptide mitogen, insulin-like growth factor II (IGF-II). The IGF-II-binding site has been mapped to repeat 11 of the EC region, with high affinity binding being conferred by residues contributed by the 13th repeat (23Dahms N.M. Wick D.A. Brzycki-Wessell M.A. J. Biol. Chem. 1994; 269: 3802-3809Abstract Full Text PDF PubMed Google Scholar, 24Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar, 25Schmidt B. Kiecke-Siemsen C. Waheed A. Braulke T. von Figura K. J. Biol. Chem. 1995; 270: 14975-14982Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 26Devi G.R. Byrd J.C. Slentz D.H. MacDonald R.G. Mol. Endocrinol. 1998; 12: 1661-1672Crossref PubMed Scopus (51) Google Scholar). Repeat 13 is thought to act as an enhancer of IGF-II affinity by slowing the rate of IGF-II dissociation (27Linnell J. Groeger G. Hassan A.B. J. Biol. Chem. 2001; 276: 23986-23991Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Structural analyses of repeat 11 identified the putative IGF-II-binding site in a hydrophobic pocket at the end of a β-barrel structure (28Brown J. Esnouf R.M. Jones M.A. Linnell J. Harlos K. Hassan A.B. Jones E.Y. EMBO J. 2002; 21: 1054-1062Crossref PubMed Scopus (89) Google Scholar). Another member of this class is retinoic acid, a unique ligand for the M6P/IGF2R in that it binds the cytoplasmic region and is thought to function by altering intracellular trafficking of the M6P/IGF2R and its cargo (29Kang J.X. Li Y. Leaf A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13671-13676Crossref PubMed Scopus (117) Google Scholar). The other members of the non-Man-6-P-containing ligands are urokinase-type plasminogen activator receptor (uPAR) and plasminogen, whose binding sites have been mapped to a peptide region within EC repeat 1 (30Nykjær A. Christensen E.I. Vorum H. Hager H. Petersen C.M. Røigaard H. Min H.Y. Vilhardt F. Møller L.B. Kornfeld S. Gliemann J. J. Cell Biol. 1998; 141: 815-828Crossref PubMed Scopus (133) Google Scholar, 31Godár S. Horejsi V. Weidle U.H. Binder B.R. Hansmann C. Stockinger H. Eur. J. Immunol. 1999; 29: 1004-1013Crossref PubMed Scopus (148) Google Scholar, 32Leksa V. Godár S. Cebecauer M. Hilgert I. Breuss J. Weidle U.H. Horejsi V. Binder B.R. Stockinger H. J. Biol. Chem. 2002; 277: 40575-40582Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The proposed function for the interactions between the M6P/IGF2R and these ligands is involvement in the complex responsible for the activation of TGF-β1 at the cell surface, as well as endocytosis and targeting of uPAR for degradation (31Godár S. Horejsi V. Weidle U.H. Binder B.R. Hansmann C. Stockinger H. Eur. J. Immunol. 1999; 29: 1004-1013Crossref PubMed Scopus (148) Google Scholar, 32Leksa V. Godár S. Cebecauer M. Hilgert I. Breuss J. Weidle U.H. Horejsi V. Binder B.R. Stockinger H. J. Biol. Chem. 2002; 277: 40575-40582Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The uPAR-M6P/IGF2R interaction appears to be weak, of low affinity, and confined to a small subpopulation of uPAR molecules (33Kreiling J.L. Byrd J.C. Deisz R.J. Mizukami I.F. Todd III, R.F. MacDonald R.G. J. Biol. Chem. 2003; 278: 20628-20637Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), which calls into question the physiological relevance of this interaction.Recent crystal structure data have given insight into structural features of the M6P/IGF2R (28Brown J. Esnouf R.M. Jones M.A. Linnell J. Harlos K. Hassan A.B. Jones E.Y. EMBO J. 2002; 21: 1054-1062Crossref PubMed Scopus (89) Google Scholar, 34Olson L.J. Yammani R.D. Dahms N.M. Kim J.J. EMBO J. 2004; 23: 2019-2028Crossref PubMed Scopus (55) Google Scholar). The crystal structures for repeat 11 by Brown et al. (28Brown J. Esnouf R.M. Jones M.A. Linnell J. Harlos K. Hassan A.B. Jones E.Y. EMBO J. 2002; 21: 1054-1062Crossref PubMed Scopus (89) Google Scholar) and repeats 1–3 by Olson et al. (34Olson L.J. Yammani R.D. Dahms N.M. Kim J.J. EMBO J. 2004; 23: 2019-2028Crossref PubMed Scopus (55) Google Scholar, 35Olson L.J. Dahms N.M. Kim J.J. J. Biol. Chem. 2004; 279: 34000-34009Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) have allowed these groups to propose different models for the overall structure of the EC domain of the M6P/IGF2R. The EC domain of the receptor shows considerable homology among repeats and the cation-dependent Man-6-P-receptor (16–38% identity) (4Lobel P. Dahms N.M. Kornfeld S. J. Biol. Chem. 1988; 263: 2563-2570Abstract Full Text PDF PubMed Google Scholar). This high level of sequence identity accounts for structural similarities among domains, including conserved disulfide bond organization, random coil linker regions connecting the domains, and an overall core flattened β-barrel structure. The 1–3 triple-repeat crystal revealed a structure in which repeat 3 sits on top of repeats 1 and 2 (34Olson L.J. Yammani R.D. Dahms N.M. Kim J.J. EMBO J. 2004; 23: 2019-2028Crossref PubMed Scopus (55) Google Scholar). Olson et al. (34Olson L.J. Yammani R.D. Dahms N.M. Kim J.J. EMBO J. 2004; 23: 2019-2028Crossref PubMed Scopus (55) Google Scholar) have proposed that the M6P/IGF2R forms distinct structural units for every three repeats of the EC region, producing five tri-repeat units that stack in a back-to-front manner. In this model, the IGF-II-binding site is located on the opposite face of the structure relative to the Man-6-P-binding sites.Traditionally thought to function as a monomer (36Perdue J.F. Chan J.K. Thibault C. Radaj P. Mills B. Daughaday W.H. J. Biol. Chem. 1983; 258: 7800-7811Abstract Full Text PDF PubMed Google Scholar), the M6P/IGF2R is now considered to operate optimally in the membrane as an oligomer for high affinity Man-6-P binding and efficient internalization of ligands (37York S.J. Arneson L.S. Gregory W.T. Dahms N.M. Kornfeld S. J. Biol. Chem. 1999; 274: 1164-1171Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 38Byrd J.C. MacDonald R.G. J. Biol. Chem. 2000; 275: 18638-18646Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 39Byrd J.C. Park J.H. Schaffer B.S. Garmroudi F. MacDonald R.G. J. Biol. Chem. 2000; 275: 18647-18656Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Intermolecular cross-linking of two M6P/IGF2R partners was shown to occur upon binding of the multivalent ligand, β-glucuronidase, resulting in increased rate of ligand internalization (37York S.J. Arneson L.S. Gregory W.T. Dahms N.M. Kornfeld S. J. Biol. Chem. 1999; 274: 1164-1171Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The initial rate of internalization of β-glucuronidase was faster than for the monovalent ligand, IGF-II, which showed that multivalent ligands enhance the rate of receptor movement, likely due to clustering of the M6P/IGF2R for improved interaction with the endocytic machinery in the formation of clathrin-coated pits (37York S.J. Arneson L.S. Gregory W.T. Dahms N.M. Kornfeld S. J. Biol. Chem. 1999; 274: 1164-1171Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Further studies demonstrated that alignment of the Man-6-P binding domains of monomeric partners of a receptor dimer is responsible for bivalent, high affinity binding, also supporting the importance of receptor oligomerization (38Byrd J.C. MacDonald R.G. J. Biol. Chem. 2000; 275: 18638-18646Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).In order to determine the interrelationship between dimer formation and the function of the ligand binding domains of the receptor, we co-expressed full-length receptors with truncated receptors from which either the N- or C-terminal EC repeats were deleted and that lacked functional ligand binding domains and/or transmembrane and cytoplasmic domains. One of the main goals of this project was to map the dimerization domain(s) of the M6P/IGF2R. We hypothesized that one or more dimer interaction domains would be located at or near the ligand binding domains in the EC region of the receptor, and that these regions would contribute preferentially to receptor dimerization. A panel of M6P/IGF2R minireceptors was constructed to test for association with a full-length version of the receptor. It was observed that all of the truncated receptors were able to associate with the full-length receptor, suggesting that dimerization domains or contacts occur all along the EC region of the receptor, not just near regions of ligand interaction. However, repeat 12 seems to be particularly important to association, as N-terminally truncated minireceptors lacking this repeat gave distinctive results in the immunoprecipitation assays. We conclude that a distinct dimerization domain for the M6P/IGF2R does not exist per se, but instead, interactions between monomeric receptor partners apparently occur all along the EC region of the receptor with special contribution made by repeat 12.EXPERIMENTAL PROCEDURESMaterialsOligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA) or the University of Nebraska Medical Center Molecular Biology Core Facility (Omaha, NE). d-Man-6-P, disodium salt, anti-(α)-FLAG M2 antibody, α-FLAG M2-agarose affinity gel, and the bicinchoninic acid kit for protein determination were purchased from Sigma. The α-Myc 9E10 antibody was purchased from Upstate Biotechnology, Inc., or the University of Nebraska Medical Center Monoclonal Antibody Facility (Omaha, NE). The polyclonal α-13D antibody (referred to as α-M6P/IGF2R throughout this report) that recognizes a peptide domain in repeat 4 of the M6P/IGF2R has been described previously (40MacDonald R.G. Tepper M.A. Clairmont K.B. Perregaux S.B. Czech M.P. J. Biol. Chem. 1989; 264: 3256-3261Abstract Full Text PDF PubMed Google Scholar). Rabbit α-mouse IgG was from Dako (Carpinteria, CA). Carrier-free Na125I and 125I-protein A were from PerkinElmer Life Sciences. Recombinant human IGFs were provided by M. H. Niedenthal (Lilly). Radiolabeled IGF-II and unlabeled and radiolabeled pentamannose phosphate-bovine serum albumin (PMP-BSA) were prepared as described previously (41Byrd J.C. Devi G.R. de Souza A.T. Jirtle R.L. MacDonald R.G. J. Biol. Chem. 1999; 274: 24408-24416Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The pCMV5 vector was provided by Dr. David W. Russell (University of Texas Southwestern Medical Center, Dallas, TX) (42Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar). The 8.6-bp human M6P/IGF2R cDNA and affinity-purified human β-glucuronidase (hGUS) were provided by Dr. William S. Sly (St. Louis University Medical Center, St. Louis, MO) (3Oshima A. Nolan C.M. Kyle J.W. Grubb J.H. Sly W.S. J. Biol. Chem. 1988; 263: 2553-2562Abstract Full Text PDF PubMed Google Scholar). Radiolabeled hGUS was prepared by iodination using precoated IODO-GEN tubes (Pierce) according to the manufacturer's specifications to a specific activity of 26–40 Ci/g. Other reagents and supplies were obtained from sources as indicated.Preparation, Expression, and Analysis of Epitope-tagged MinireceptorsThe truncated M6P/IGF2R minireceptors 1-8F, 1-9F, 1-9F-R/A, 1-11F, and 1-15F were tagged with the eight-residue FLAG epitope (DYKDDDDK) followed by a stop codon and an XbaI restriction site at the C terminus and cloned into the pCMV5 vector as described previously (Fig. 1A) (38Byrd J.C. MacDonald R.G. J. Biol. Chem. 2000; 275: 18638-18646Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). By using the full-length M6P/IGF2R cDNA as the template, the following minireceptors, containing the EC repeats starting with the repeat indicated in the name of the receptor and ending with the 15th repeat, followed by the transmembrane and cytoplasmic regions of the M6P/IGF2R, were synthesized by amplification with Vent™ polymerase (New England Biolabs, Beverly, MA): 10-15CF (nt 4239–7620), 11-15CF (nt 4675–7620), 12-15CF (nt 5092–7620), and 13-15CF (nt 5542–7620) (Fig. 5A). To ensure consistent translation, the signal sequence containing the N-terminal 71 residues of repeat 1 was fused to the beginning of each construct as described previously (43Garmroudi F. Devi G. Slentz D.H. Schaffer B.S. MacDonald R.G. Mol. Endocrinol. 1996; 10: 642-651Crossref PubMed Google Scholar). All of these minireceptors were C-terminally tagged with the FLAG epitope followed by a stop codon and XbaI restriction site for cloning purposes.Fig. 5Ligand blot analysis of the N-terminally truncated M6P/IGF2R minireceptors. Proteins from aliquots (25 μl) of Triton X-100 extracts from 293T cells transfected with the N-terminally truncated minireceptors were treated as in the legend to Fig. 2. A, 1 × 106 cpm 125I-IGF-II; or B, 1 × 106 cpm PMP-BSA. The blots were exposed to film by autoradiography. The arrows indicate endogenous M6P/IGF2R binding to the radioligands.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The cDNA plasmids in the vector pCMV5R1X encoding the 10-15CF-I/T and 11-15CF-I/T minireceptors, bearing the isoleucine to threonine mutation that has been shown previously to prevent IGF-II binding to the M6P/IGF2R, were synthesized from the 10-15CF and 11-15CF constructs as well as a 1-15F construct containing the I1572T mutation (1-15F-I/T) (39Byrd J.C. Park J.H. Schaffer B.S. Garmroudi F. MacDonald R.G. J. Biol. Chem. 2000; 275: 18647-18656Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, the 10-15CF or 11-15CF and 1-15F-I/T cDNAs were digested with BstEII (M6P/IGF2R nt 4698) and BstBI (M6P/IGF2R nt 5507) serially, and the resulting fragments of interest (809-bp fragment for 1-15F-I/T, corresponding to the region containing the Ile → Thr mutation, and the larger fragment (>7000 bp) for 10-15CF and 11-15CF, which also encompasses the pCMV5 plasmid), were gel-purified (Qiagen, Valencia, CA). The purified fragments were then ligated together using T4 DNA Ligase (Invitrogen) and used to transform XL-10 Gold competent cells (Stratagene, La Jolla, CA).The full-length Myc-tagged M6P/IGF2R was prepared as follows: full-length M6P/IGF2R cDNA that had been digested with EagI and re-ligated (lacking nt 162–5319) was used as template for amplification with a 5′-primer containing an XhoI restriction site preceding the sequence corresponding to nt 94–113 of the receptor cDNA and a 3′-primer with sequence complementary to nt 7602–7620 at the C-terminal end of the expressed M6P/IGF2R followed by the 36-nt sequence encoding the Myc epitope, MEQKLISEEDLN (44Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2154) Google Scholar), followed by two stop codons and an XbaI site. The products from these amplifications were digested with XbaI and XhoI and subcloned into pBKCMV (Invitrogen). These plasmids were then digested with HindIII and XbaI and subcloned into the target vector, pCMV5. Finally, wild-type EagI fragments were subcloned into the construct, reconstituting the completed M6P/IGF2R-Myc (WT-M) cDNA construct.A full-length, Myc-tagged M6P/IGF2R triple mutant (R2AxI/T-M) for residues in the repeats 3 and 9 Man-6-P binding domains (R426A and R1325A, respectively) and the repeat 11 IGF-II binding domain (I1572T) was prepared as follows: nt 100–3242 of the M6P/IGF2R cDNA served as template for amplification using a 5′-primer containing an EcoRI restriction site preceding the sequence corresponding to nt 1112–1129 of the receptor cDNA and a 3′-primer with sequence complementary to nt 2474–2487 of the M6P/IGF2R cDNA, followed by an XhoI site. The products from these amplifications were digested with EcoRI and XhoI and subcloned into pBluescript SK II+ (pBSKII+) (Stratagene). This construct was subjected to two rounds of amplification with primers designed to incorporate the R426A mutation responsible for altering the EC repeat 3 Man-6-P-binding site using the Megaprimer approach (45Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). The first round of amplification involved producing the mutation, by amplifying from that site (nt 1425) to the 3′ end of the minireceptor (nt 2487). This "megaprimer" was then used in a second round of amplification with the 5′-primer used above. The repeat 3 mutant amplification product was digested with EcoRI and XhoI and subcloned back into pBSKII+. An ∼1-kb BsmI-BsmI fragment (sites at M6P/IGF2R nt 1408 and 2449) containing the mutation was removed from the megaprimer and subcloned into the corresponding positions of pBSKII+/Kpn, which contained a 2.2-kb KpnI-KpnI fragment derived from M6P/IGF2R nt 100–3242, creating a pBSKII+/Kpn-R426A minireceptor with the 3rd repeat Man-6-P-binding mutation. The KpnI-KpnI fragment from this construct was then subcloned into pCMV5/R1325A-M, a full-length Myc-tagged receptor containing the 9th repeat Man-6-P-binding mutation synthesized as described previously (39Byrd J.C. Park J.H. Schaffer B.S. Garmroudi F. MacDonald R.G. J. Biol. Chem. 2000; 275: 18647-18656Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), that had also been digested with KpnI and XmnI, creating a full-length M6P/IGF2R bearing Man-6-P-binding site mutations at both repeats 3 and 9 (R2A-M). The R2A-M cDNA was digested with AflII (M6P/IGF2R nt 4740) and MluI (pCMV5 nt 933), and this ∼5-kb fragment was subcloned into pCMV5/I1572T-M, a full-length Myc-tagged receptor containing the 11th repeat IGF-II-binding mutation at nt 4862 (24Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar), which had also been digested with AflII and MluI. The final product resulted in the binding-defective triple mutant, R2AxI/T-M, in the vector pCMV5.Transient expression of the minireceptors by calcium phosphate-mediated transfection into 293T human embryonic kidney cells and immunoblot analysis of cell lysates to measure expression of the truncated and full-length receptors were performed as described previously (39Byrd J.C. Park J.H. Schaffer B.S. Garmroudi F. MacDonald R.G. J. Biol. Chem. 2000; 275: 18647-18656Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 46Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2278) Google Scholar). Additionally, expression was tested by the use of the α-M6P/IGF2R polyclonal antibody (40MacDonald R.G. Tepper M.A. Clairmont K.B. Perregaux S.B. Czech M.P. J. Biol. Chem. 1989; 264: 3256-3261Abstract Full Text PDF PubMed Google Scholar). Aliquots (25 μl) of Triton X-100 extracts were resolved by electrophoresis on 6% reducing SDS-polyacrylamide gels in sample buffer (50 m
Referência(s)