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The Rate of Internalization of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is Enhanced by Multivalent Ligand Binding

1999; Elsevier BV; Volume: 274; Issue: 2 Linguagem: Inglês

10.1074/jbc.274.2.1164

ISSN

1083-351X

Autores

Sally J. York, Lynne S. Arneson, Walter Gregory, Nancy Dahms, Stuart Kornfeld,

Tópico(s)

Growth Hormone and Insulin-like Growth Factors

Resumo

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, β-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that β-glucuronidase entered the cell ∼3–4-fold faster than IGF-II. Unlabeled β-glucuronidase stimulated the rate of internalization of125I-IGF-II to equal that of125I-β-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize β-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with β-glucuronidase generated a complex composed of two receptors and one β-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization. The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, β-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that β-glucuronidase entered the cell ∼3–4-fold faster than IGF-II. Unlabeled β-glucuronidase stimulated the rate of internalization of125I-IGF-II to equal that of125I-β-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize β-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with β-glucuronidase generated a complex composed of two receptors and one β-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization. The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) 1The abbreviations used are: M6P/IGF-II receptor, mannose 6-phosphate/insulin-like growth factor II receptor; IGF-II, insulin-like growth factor II; Man-6-P, mannose 6-phosphate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FPLC, fast protein liquid chromatography. is a type I transmembrane glycoprotein that cycles through the Golgi, endosomes, and the plasma membrane to carry out its role in the biogenesis of lysosomes and in the clearance of the polypeptide insulin-like growth factor II (IGF-II) (1Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (949) Google Scholar, 2Hille-Rehfeld A. Biochim. Biophys. Acta. 1995; 1241: 177-194Crossref PubMed Scopus (218) Google Scholar). In the Golgi, the receptor binds newly synthesized acid hydrolases modified with mannose 6-phosphate (Man-6-P) residues on their asparagine-linked oligosaccharides and transports them to endosomes via clathrin-coated vesicles (3Kyle J.W. Nolan C.M. Oshima A. Sly W.S. J. Biol. Chem. 1988; 263: 16230-16235Abstract Full Text PDF PubMed Google Scholar, 4Geuze H.J. Slot J.W. Strous G.J. Hasilik A. von Figura K. J. Cell Biol. 1985; 101: 2253-2262Crossref PubMed Scopus (164) Google Scholar, 5Lobel P. Fujimoto K. Ye R.D. Griffiths G. Kornfeld S. Cell. 1989; 57: 787-796Abstract Full Text PDF PubMed Scopus (208) Google Scholar). The acid hydrolases are released in the acidified endosome and then packaged into lysosomes while the receptor either returns to the Golgi to bind another ligand or moves to the plasma membrane (6Duncan J.R. Kornfeld S. J. Cell Biol. 1988; 106: 617-628Crossref PubMed Scopus (180) Google Scholar, 7Jin M. Sahagian G.G. Snider M.D. J. Biol. Chem. 1989; 264: 7675-7680Abstract Full Text PDF PubMed Google Scholar). At the plasma membrane, the M6P/IGF-II receptor mediates internalization of Man-6-P-containing ligands and IGF-II (3Kyle J.W. Nolan C.M. Oshima A. Sly W.S. J. Biol. Chem. 1988; 263: 16230-16235Abstract Full Text PDF PubMed Google Scholar,5Lobel P. Fujimoto K. Ye R.D. Griffiths G. Kornfeld S. Cell. 1989; 57: 787-796Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 8Kiess W. Blickenstaff G.D. Sklar M.M. Thomas C.L. Nissley S.P. Sahagian G.G. J. Biol. Chem. 1988; 263: 9339-9344Abstract Full Text PDF PubMed Google Scholar). The interactions of IGF-II and Man-6-P-containing ligands with the M6P/IGF-II receptor have been characterized in several studies (8Kiess W. Blickenstaff G.D. Sklar M.M. Thomas C.L. Nissley S.P. Sahagian G.G. J. Biol. Chem. 1988; 263: 9339-9344Abstract Full Text PDF PubMed Google Scholar, 9Kiess W. Thomas C.L. Greenstein L.A. Lee L. Sklar M.M. Rechler M.M. Sahagian G.G. Nissley S.P. J. Biol. Chem. 1989; 264: 4710-4714Abstract Full Text PDF PubMed Google Scholar, 10MacDonald R.G. Pfeffer S.R. Coussens L. Tepper M.A. Brocklebank C.M. Mole J.E. Anderson J.K. Chen E. Czech M.P. Ullrich A. Science. 1988; 239: 1134-1137Crossref PubMed Scopus (276) Google Scholar, 11Morgan D.O. Edman J.C. Standring D.N. Fried V.A. Smith M.C. Roth R.A. Rutter W.J. Nature. 1987; 329: 301-307Crossref PubMed Scopus (669) Google Scholar, 12Tong P.Y. Tollefsen S.E. Kornfeld S. J. Biol. Chem. 1988; 263: 2585-2588Abstract Full Text PDF PubMed Google Scholar). The extracellular portion of the M6P/IGF-II receptor contains 15 homologous repeating domains of ∼147 amino acids each (13Lobel P. Dahms N.M. Kornfeld S. J. Biol. Chem. 1988; 263: 2563-2570Abstract Full Text PDF PubMed Google Scholar). Domains 3 and 9 (numbering from the amino terminus) each bind 1 mol of Man-6-P, and the single IGF-II-binding site has been mapped to domain 11 in the extracellular region (14Dahms 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, 15Garmroudi F. MacDonald R.G. J. Biol. Chem. 1994; 269: 26944-26952Abstract Full Text PDF PubMed Google Scholar, 16Schmidt 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 (76) Google Scholar). Man-6-P residues do not inhibit binding of IGF-II to the receptor, verifying that the two ligand-binding sites are distinct. However, proteins containing Man-6-P residues do compete with IGF-II for receptor binding, and IGF-II can inhibit binding of lysosomal enzymes to the receptor (8Kiess W. Blickenstaff G.D. Sklar M.M. Thomas C.L. Nissley S.P. Sahagian G.G. J. Biol. Chem. 1988; 263: 9339-9344Abstract Full Text PDF PubMed Google Scholar, 9Kiess W. Thomas C.L. Greenstein L.A. Lee L. Sklar M.M. Rechler M.M. Sahagian G.G. Nissley S.P. J. Biol. Chem. 1989; 264: 4710-4714Abstract Full Text PDF PubMed Google Scholar, 10MacDonald R.G. Pfeffer S.R. Coussens L. Tepper M.A. Brocklebank C.M. Mole J.E. Anderson J.K. Chen E. Czech M.P. Ullrich A. Science. 1988; 239: 1134-1137Crossref PubMed Scopus (276) Google Scholar, 17Kiess W. Thomas C.L. Sklar M.M. Nissley S.P. Eur. J. Biochem. 1990; 190: 71-77Crossref PubMed Scopus (48) Google Scholar). In neither case is the competition complete, and the most plausible explanation is that the inhibition is due to steric hindrance. Although the M6P/IGF-II receptor has been shown to be constitutively internalized from the cell surface, it is not clear whether ligand binding influences the trafficking of the receptor. It has been reported that in the absence of ligand, the M6P/IGF-II receptor accumulates in the Golgi, whereas the addition of lysosomotropic agents that prevent the release of ligand from the receptor in endosomes results in an accumulation of the receptor in these organelles (18Brown W.J. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5135-5139Crossref PubMed Scopus (42) Google Scholar, 19Brown W.J. Constantinescu E. Farquhar M.G. J. Cell Biol. 1984; 99: 320-326Crossref PubMed Scopus (63) Google Scholar, 20Brown W.J. Goodhouse J. Farquhar M.G. J. Cell Biol. 1986; 103: 1235-1247Crossref PubMed Scopus (195) Google Scholar). Other investigators have found that constitutive trafficking of the M6P/IGF-II receptor continues under these conditions (21Braulke T. Gartung C. Hasilik A. von Figura K. J. Cell Biol. 1987; 104: 1735-1742Crossref PubMed Scopus (85) Google Scholar, 22Pfeffer S.R. J. Cell Biol. 1987; 105: 229-234Crossref PubMed Scopus (35) Google Scholar). Together, these data are consistent with the concept that ligand binding modulates the rate of receptor trafficking. Thus, the absence or presence of bound ligand may regulate the trafficking from specific compartments, resulting in a shift in the steady-state distribution of the receptor. However, none of these studies have actually determined the kinetics of receptor trafficking. In this study, we have compared the internalization of β-glucuronidase, a homotetramer with multiple phosphorylated oligosaccharides, with that of IGF-II. We found that the initial rate of internalization of β-glucuronidase is much more rapid than that of IGF-II, providing direct evidence that a multivalent ligand enhances the rate of movement of the receptor. Furthermore, we present data that the mechanism of this effect is due to dimerization of the receptor. Recombinant human IGF-II was purchased from Bachem California; IGF-II-(del 1–6) from Upstate Biotechnology, Inc., Na125I from Amersham Pharmacia Biotech; lactoperoxidase from Calbiochem; Man-6-P from Sigma; and Lipofectin and G418 from Life Technologies, Inc. Other reagent-grade chemicals were from standard suppliers. The bivalent ligand (Ac-Thr-[α-d-Man-6-P(α1–2)α-d-Man]-Lys-(ABz)-Thr-[α-d-Man-6-P(α1–2)α-d-Man]-NH2) was kindly provided by Dr. K. Bock (Carlsberg Laboratory, Copenhagen, Denmark) (23Franzyk H. Christensen M.K. Jorgensen R.M. Meldal M. Cordes H. Mouritsen S. Bock K. Bioorg. Med. Chem. 1997; 5: 21-40Crossref PubMed Scopus (35) Google Scholar). The transfection of the M6P/IGF-II receptor-negative mouse L cell line (L(Rec−)) with constructs encoding the wild-type receptor and a mutant receptor with a 29-amino acid cytoplasmic tail to give the Cc2 and 344 cell lines, respectively, has been previously described (5Lobel P. Fujimoto K. Ye R.D. Griffiths G. Kornfeld S. Cell. 1989; 57: 787-796Abstract Full Text PDF PubMed Scopus (208) Google Scholar,24Jadot M. Canfield W.M. Gregory W. Kornfeld S. J. Biol. Chem. 1992; 267: 11069-11077Abstract Full Text PDF PubMed Google Scholar). The Dom3ala cell line expressing a receptor with an R435A mutation was generated as described (25Marron-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). Human β-glucuronidase was purified from the secretions of 13.2.1 mouse L cells as described previously (24Jadot M. Canfield W.M. Gregory W. Kornfeld S. J. Biol. Chem. 1992; 267: 11069-11077Abstract Full Text PDF PubMed Google Scholar). This cell line, which has been engineered to secrete large amounts of β-glucuronidase, was kindly provided by Dr. William Sly (St. Louis University). For iodination, 30 μg of human β-glucuronidase was brought to 500 μl in PBS (pH 7.4); 2.5 μl of 1.86 μm lactoperoxidase, 1 mCi of Na125I, and 2 μl of 0.5 nm hydrogen peroxide were added to start the reaction. After 3 min at room temperature, another 2 μl of 0.5 nm hydrogen peroxide was added, and after an additional 3 min, the reaction was stopped by adding 200 μl of quench solution (1 m NaCl, 100 mm NaI, 50 mm NaPO4 (pH 7.5), 1 mmNaN3, 2 mg/ml BSA, and 1 mg/ml protamine sulfate). The quenched reaction mixture was loaded onto a 1-ml M6P/IGF-II receptor affinity column (26Hoflack B. Fujimoto K. Kornfeld S. J. Biol. Chem. 1987; 262: 123-129Abstract Full Text PDF PubMed Google Scholar) equilibrated in PBS and 0.1% BSA. After extensive washing, the β-glucuronidase was eluted with PBS and 0.1% BSA containing 10 mm Man-6-P. The peak fractions were pooled and dialyzed against PBS to remove the Man-6-P, and the125I-β-glucuronidase was stored at 4 °C. The typical specific activity assuming complete protein recovery was 106 to 107 cpm/μg of protein. 10 μg of IGF-II was iodinated using the lactoperoxidase method described for β-glucuronidase. After quenching, the reaction mixture was loaded onto a Sephadex G-25 column equilibrated in PBS and 0.1% BSA. Three peaks of radioactivity were observed and subjected to trichloroacetic acid precipitation. The second peak was found to be 98% trichloroacetic acid-precipitable and contained monomeric IGF-II. The iodinated ligand was stored at 4 °C. The typical specific activity assuming complete protein recovery was 107 cpm/μg of protein. IGF-II-(del 1–6) was iodinated by coating an Eppendorf tube with 50 μg of IODO-GEN (Pierce) and adding 5 μg of IGF-II-(del 1–6) and 1 mCi of Na125I. The mixture was incubated at room temperature for 3 min and then loaded onto a PD10 column (Amersham Pharmacia Biotech) pre-equilibrated in PBS plus 1% BSA. Fractions containing the first peak were pooled and stored at 4 °C. The typical activity assuming complete protein recovery was 108 cpm/μg of protein. Cells were grown to confluence in 12-well plates. The cells were rinsed twice in ice-cold PBS and 1% BSA, and ligand was added in cold α-minimal Eagle's medium and 2% BSA (0.5 ml/well). 125I-IGF-II was added to a 2 nm final concentration, and125I-β-glucuronidase was typically added to a final concentration of 0.12 nm. The plates were then floated on an ice water bath for 30 min. Unbound ligand was removed, and the wells were rapidly washed five times with 1 ml of ice-cold PBS and 1% BSA. To the three wells used for the 0 time point was added 1 ml of ice-cold stop/strip solution (SSS; 0.2 m acetic acid (pH 3.5) and 0.5 m NaCl). The plate was then floated in a 37 °C water bath, and 0.5 ml of α-minimal Eagle's medium prewarmed to 37 °C was quickly added to the remaining wells to initiate internalization. At each stopping point, the α-minimal Eagle's medium in the well was removed to a tube for counting, and 1 ml of cold SSS was added. At the end of the experiment (usually 3 min), the plate was removed from the water bath, and the surface-bound ligand was stripped from each well by incubation for a total of 10 min in ice-cold SSS (1 ml for 5 min, twice) and counted. The cells were then solubilized in 0.1n NaOH (1 ml, twice) and counted. The M6P/IGF-II receptor-negative cell line was used as a control for non-receptor binding of 125I-IGF-II and125I-β-glucuronidase. The sum of the labeled ligand remaining on the cell surface at the end of the internalization experiment (receptor-bound ligand) and the internalized ligand was used as a measure of the maximum potential internalization. The fraction of this value internalized at each time point was calculated and plotted. This calculation method was used to exclude the contributions of a specific, non-M6P/IGF-II receptor binding site for IGF-II observed in all the cell lines, including the receptor-negative cells. This site has a lower affinity for IGF-II than the M6P/IGF-II receptor, and with incubation at 37 °C, the ligand is released into the medium rather than internalized. This calculation method was used for both IGF-II and β-glucuronidase binding studies. 110 g of fresh bovine liver was minced and blended in a Waring blender in 200 ml of ice-cold extraction buffer (50 mm imidazole (pH 7), 150 mm NaCl, 5 mm sodium β-glycerophosphate, 2% Triton X-100, 0.25% deoxycholate, 10 mm EDTA, 50 μg/ml leupeptin, 50 μg/ml aprotinin, 50 μg/ml trypsin inhibitor, and 50 μg/ml phenylmethylsulfonyl fluoride) for ∼10 s, four times. The homogenate was centrifuged at 30,000 × g for 30 min, and the supernatant was poured through cheesecloth. A ∼15-ml packed volume of phosphopentamannose-agarose beads (27Distler J.J. Guo J. Jourdian G.W. Srivastava O.P. Hindsgaul O. J. Biol. Chem. 1991; 266: 21687-21692Abstract Full Text PDF PubMed Google Scholar) was washed with extraction buffer without protease inhibitors and added to the supernatant. Receptor binding was allowed to occur for 30 min at 4 °C while rocking. The beads were collected by filtering over a coarse Buchner funnel, and the agarose beads were washed with 1 liter of extraction buffer followed by 500 ml of wash buffer (50 mm imidazole (pH 7), 150 mm NaCl, 5 mm sodium β-glycerophosphate, and 0.05% Triton X-100). The washed agarose was poured into a column, and the M6P/IGF-II receptor was eluted with wash buffer containing 10 mmMan-6-P. Fractions containing the receptor were pooled, and protein concentration was determined by the Bradford assay (47Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222135) Google Scholar). The recovery of the receptor was 660 μg. A Superose 6 FPLC column was equilibrated in filtered and degassed wash buffer. Protein standards were run and detected by absorbance at 280 nm. TheK d, defined as (V e −V o)/(V t −V o), was determined, and theErf −1 (1 − K d) was plotted versus the known Stokes radius of the protein standards (28Ackers G.K. J. Biol. Chem. 1967; 242: 3237-3238Abstract Full Text PDF Google Scholar). The V e of the membrane form of the M6P/IGF-II receptor was determined by collecting 1-ml fractions and analyzing the contents by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining of the gel. The V e of complexes containing β-glucuronidase was determined by β-glucuronidase assays. Continuous 6–21% sucrose gradients (4.8 ml) were prepared in wash buffer and allowed to equilibrate at 4 °C for 1 h. Samples were loaded onto the gradients, and the gradients were centrifuged for 4 h at 237,000 × g av in an SW 55Ti rotor. Fractions (240 or 120 μl) were collected from the top of the gradient. Refractive indices of each fraction were measured to determine linearity of the gradients. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis following chloroform/methanol precipitation or by β-glucuronidase assays as described below. The sedimentation coefficients of the proteins and complexes were determined using protein standards as markers (29Sadler J.E. Rearick J.I. Paulson J.C. Hill R.L. J. Biol. Chem. 1979; 254: 4434-4443Abstract Full Text PDF PubMed Google Scholar). A sample of each fraction to be tested (2–10 μl) was incubated with 100 μl of 10 mm4-methylumbelliferyl β-d-glucuronide (Sigma) suspended in 0.1 m sodium acetate (pH 5.0) at 37 °C for 10–60 min. Following incubation, 3 ml of 0.25 m glycine (pH 10.3) was added to stop the reaction, and the fluorescence was determined in a Turner fluorometer. The internalization rates of IGF-II and β-glucuronidase were compared using 125I-IGF-II and125I-β-glucuronidase in an adaptation of the endocytosis assay developed by Jadot et al. (24Jadot M. Canfield W.M. Gregory W. Kornfeld S. J. Biol. Chem. 1992; 267: 11069-11077Abstract Full Text PDF PubMed Google Scholar). Following an initial lag of 15–20 s, 125I-IGF-II was internalized in a nearly linear fashion, with a t12 of 2–3 min (Fig.1 B). No plateau was observed during the 5-min incubation because very little of the IGF-II was released from the M6P/IGF-II receptor during the course of this assay. The internalization of IGF-II occurred exclusively via the M6P/IGF-II receptor since the untransfected parent cell line did not take up any IGF-II under these conditions (data not shown). By contrast, β-glucuronidase was internalized ∼3–4-fold more rapidly, with a t12 of 30–45 s (Fig.1 A). A plateau was reached when essentially all of the ligand originally present on the cell had been either internalized or released from the receptor into the medium, where it was greatly diluted. Together, these data show that β-glucuronidase binding stimulates the rate of receptor internalization over that observed upon IGF-II binding. To test whether the increased rate of internalization of the receptor with bound β-glucuronidase was a result of ligand occupation of the two Man-6-P-binding sites, the effect of 10 mm Man-6-P on the rate of 125I-IGF-II uptake was determined. This concentration of Man-6-P saturated the Man-6-P-binding sites on the receptor. Although Man-6-P caused a small increase in total125I-IGF-II binding, it had no effect on the rate of125I-IGF-II internalization (data not shown), indicating that the increase in internalization rate was not solely due to Man-6-P binding. The effect of β-glucuronidase on the internalization of125I-IGF-II was next determined. In this experiment, the simultaneous binding of 125I-IGF-II and β-glucuronidase was maximized by first incubating cells on ice with125I-IGF-II for 5 min to allow maximum binding of this ligand. Excess unlabeled β-glucuronidase (10 nm) was then added to each well for an additional 25 min on ice. The cells were washed, and the uptake of 125I-IGF-II was determined. The presence of β-glucuronidase stimulated the rate of endocytosis of125I-IGF-II to that observed with125I-β-glucuronidase alone (Fig.2). This indicates that the unlabeled β-glucuronidase bound to a significant fraction of the receptors that had already bound 125I-IGF-II, resulting in an increase in internalization rate that cannot be merely due to the receptor binding to Man-6-P residues. β-Glucuronidase could enhance the rate of internalization of the M6P/IGF-II receptor by promoting either intramolecular or intermolecular cross-linking. Since each monomer of the receptor contains two Man-6-P-binding sites, simultaneous binding to two Man-6-P residues on a phosphorylated oligosaccharide could induce a conformational change in the extracellular domain of the receptor that is transmitted to the cytosolic domain, where the internalization signal is located. This could result in a more favorable presentation of the internalization signal. Alternatively, the ligand could cross-link two receptor molecules, resulting in an increased density of the internalization signals. This could enhance the likelihood of the receptors being retained in a forming clathrin-coated pit, thus increasing the probability of internalization and consequently the rate. To distinguish between these possibilities, two approaches were used. First, the effect of a small bivalent Man-6-P-containing peptide on the rate of IGF-II uptake was determined. The peptide, a Thr-Lys-Thr tripeptide with a Man-6-P(α1–2)Man disaccharide attached to each threonine, has an affinity for the M6P/IGF-II receptor that is similar to that of an oligosaccharide with two Man-6-P residues and over 1000-fold higher than that of Man-6-P (23Franzyk H. Christensen M.K. Jorgensen R.M. Meldal M. Cordes H. Mouritsen S. Bock K. Bioorg. Med. Chem. 1997; 5: 21-40Crossref PubMed Scopus (35) Google Scholar, 30Tong P.Y. Gregory W. Kornfeld S. J. Biol. Chem. 1989; 264: 7962-7969Abstract Full Text PDF PubMed Google Scholar, 31Christensen M.K. Meldal M. Bock K. Cordes H. Mouritsen S. Elsner H. J. Chem. Soc. Perkin Trans. I. 1994; 1: 1299-1310Crossref Google Scholar). This high binding affinity indicates that the ligand is interacting with two binding sites on the M6P/IGF-II receptor. As shown below, this peptide does not mediate intermolecular cross-linking of the receptor. In control experiments, the peptide competed with β-glucuronidase for the Man-6-P-binding site on the receptor, but did not interfere with binding of IGF-II to the receptor (data not shown). A saturating concentration of the peptide (5 μm) did not significantly alter the rate of 125I-IGF-II internalization, whereas unlabeled β-glucuronidase accelerated the rate of125I-IGF-II uptake considerably (Fig.3). These results suggest that intramolecular cross-linking of extracellular domains 3 and 9 of the M6P/IGF-II receptor does not alter the rate of internalization. The second approach to distinguish intra- from intermolecular cross-linking utilized cells expressing a mutant receptor that has only a single functional Man-6-P-binding site. The Man-6-P binding of domain 3 was abolished by substituting an alanine for Arg-435 (14Dahms 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, 25Marron-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 full-length receptor containing this mutation was transfected into L(Rec−) cells, creating the Dom3ala cell line (25Marron-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 M6P/IGF-II receptors in this cell line are incapable of intramolecular cross-linking due to the presence of only a single functional Man-6-P-binding site per receptor, but could potentially undergo intermolecular cross-linking. The ability of Dom3ala cells to internalize β-glucuronidase and IGF-II was determined (Fig.4). In preliminary experiments, internalization of 125I-IGF-II by Dom3ala cells was partially obscured by a high background resulting from IGF-II binding to other proteins (32Francis G.L. Aplin S.E. Milner S.J. McNeil K.A. Ballard F.J. Wallace J.C. Biochem. J. 1993; 293: 713-719Crossref PubMed Scopus (75) Google Scholar). This technical problem was resolved by using IGF-II-(del 1–6), which contains a deletion that prevents binding to the IGF-II-binding proteins while maintaining internalization by the M6P/IGF-II receptor (32Francis G.L. Aplin S.E. Milner S.J. McNeil K.A. Ballard F.J. Wallace J.C. Biochem. J. 1993; 293: 713-719Crossref PubMed Scopus (75) Google Scholar). IGF-II-(del 1–6) was internalized by the wild-type M6P/IGF-II receptor at a rate similar to that observed with native IGF-II (Fig. 4 A), and the addition of unlabeled β-glucuronidase increased the rate of receptor internalization to that seen earlier (Fig. 2 A). The rate of125I-IGF-II-(del 1–6) uptake by the mutant receptor in the Dom3ala cells was similar to that of the wild-type receptor (Fig. 4 B). This was expected since neither the IGF-II-binding site nor the internalization signal in the cytoplasmic domain is different. As with wild-type receptors, the addition of unlabeled β-glucuronidase increased the rate of internalization of the mutant receptor in Dom3ala cells, as measured by125I-IGF-II-(del 1–6) uptake (Fig. 4 B). This strengthens the conclusion that intramolecular cross-linking is not responsible for the increased rate of internalization and suggests that intermolecular cross-linking mediates the enhanced internalization. The internalization rate of 125I-IGF-II-(del 1–6) by the Dom3ala cells in the presence of β-glucuronidase was not as rapid as that seen with cells expressing the wild-type receptors. This may be because the single Man-6-P-binding site on the mutant receptor results in lower binding affinity for β-glucuronidase and consequently less efficient cross-linking of receptor molecules. We next determined the state of oligomerization of purified M6P/IGF-II receptor in both the presence and absence of β-glucuronidase. Perdueet al. (33Perdue 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) have reported that the M6P/IGF-II receptor is a monomer when solubilized, whereas Stein et al. (34Stein M. Braulke T. Krentler C. Hasilik A. von Figura K. Biol. Chem. Hoppe-Seyler. 1987; 368: 937-947Crossref PubMed Scopus (59) Google Scholar) suggested that it may exist as a dimer in the plasma membrane, as determined by cross-linking studies. The M6P/IGF-II receptor was solubilized and purified from fresh bovine liver. The purified receptor was analyzed by FPLC gel filtration to determine its Stokes radius (Fig. 5, A and C) and by sedimentation in a continuous 6–21% sucrose gradient to determine its sedimentation coefficient (Fig.6). The Stokes radius of the receptor was calculated to be 79 Å, which is somewhat greater than the previously reported value of 72 Å (33Perdue 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 sedimentation coefficient was determined to be 10.1 × 1013 s, in close accordance with the previously published value (33Perdue 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 partial specific volume was calculated to be 0.73, based on the amino acid composition and expected carbohydrate additions. No corrections for detergent were applied due to the negligible amount of bound detergent found by us (compare migration relative to protein standards in H2O and D2O gradients in Fig. 6) and others (33Perdue J.F. Chan J.K. Thibault C. Radaj P. Mills B. Daughaday W.H. J. Biol

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