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• W2023262488 abstract "The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993)Endocrinology 133, 440–446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195–204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage. The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993)Endocrinology 133, 440–446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195–204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage. cation-independent mannose 6-phosphate receptor insulin-like growth factor human insulin-like growth factor recombinant human insulin-like growth factor kangaroo insulin-like growth factor reverse transcription-polymerase chain reaction SDS-polyacrylamide gel electrophoresis 10 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, and 0.005% surfactant P-20 polymerase chain reaction The cation-independent mannose 6-phosphate receptor (CI-MPR)1 is a multifunctional protein that binds proteins bearing mannose 6-phosphate moieties, as well as insulin-like growth factor (IGF)-II. IGF-II is a polypeptide mitogen related to insulin that is believed to be particularly important during placental and embryonic development (3DeChiara T.M. Efstraiadis A. Robertson E.J. Nature. 1990; 345: 78-80Crossref PubMed Scopus (1410) Google Scholar, 4Wang Z.-Q. Fung M.R. Barlow D.P. Wagner E.F. Nature. 1994; 372: 464-467Crossref PubMed Scopus (408) Google Scholar, 5Han V.K.M. Bassett N. Walton J. Challis J.R.G. J. Clin. Endocrinol. Metab. 1996; 81: 2680-2693Crossref PubMed Scopus (320) Google Scholar, 6Reynolds T.S. Stevenson K.R. Wathes D.C. Endocrinology. 1997; 138: 886-897Crossref PubMed Scopus (64) Google Scholar). The binding sites for IGF-II and mannose 6-phosphate-bearing ligands have been shown to be distinct (7Roth R.A. Stover C. Hari J. Morgan D.O. Smith M.C. Sara V. Fried V.A. Biochem. Biophys. Res. Commun. 1987; 149: 600-606Crossref PubMed Scopus (62) 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, 9Tong P.Y. Tollefsen S.E. Kornfeld S. J. Biol. Chem. 1988; 263: 2585-2588Abstract Full Text PDF PubMed Google Scholar). Whereas the role of the CI-MPR in lysosomal enzyme sorting and transport has been largely elucidated (for a review, see Ref. 10Munier-Lehmann H. Mauxion F. Hoflack B. Biochem. Soc. Trans. 1996; 24: 133-136Crossref PubMed Scopus (46) Google Scholar), the physiological role of IGF-II binding to this receptor remains unresolved and somewhat controversial. Understanding the functions of the CI-MPR in the IGF system is complicated by the presence of two other receptors, the type 1 IGF and the insulin receptors, which also bind IGF-II. Indeed, many of the effects attributed to IGF-II are mediated via the type 1 IGF receptor (11Hammerman M.R. Gavin III, J.R. J. Biol. Chem. 1984; 259: 13511-13517Abstract Full Text PDF PubMed Google Scholar, 12Zick Y. Sasaki N. Rees-Jones R.W. Grunberger G. Nissley S.P. Rechler M.M. Biochem. Biophys. Res. Commun. 1984; 119: 6-13Crossref PubMed Scopus (58) Google Scholar). However, it is widely accepted that the CI-MPR plays an important role in internalizing and degrading extracellular IGF-II (13Oka Y. Rozek L.M. Czech M.P. J. Biol. Chem. 1985; 260: 9435-9442Abstract Full Text PDF PubMed Google Scholar, 14Nolan C.M. Kyle J.W. Watanabe H. Sly W.S. Cell Regul. 1990; 1: 197-213Crossref PubMed Google Scholar, 15Polychronakos C. Piscina R. Endocrinology. 1988; 123: 2146-2148Crossref PubMed Scopus (6) Google Scholar, 16Polychronakos C. Guyda H.J. Janthly U. Posner B.I. Endocrinology. 1990; 127: 1861-1866Crossref PubMed Scopus (19) Google Scholar). On the other hand, the hypothesis that some of the biological actions of IGF-II are mediated by the CI-MPR has been difficult to prove, and much of the evidence has been contradictory. Okamoto et al. (17Okamoto T. Katada T. Murayama Y. Ui M. Ogata E. Nishimoto I. Cell. 1990; 62: 709-717Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 18Okamoto T. Ohkuni Y. Ogata E. Nishimoto I. Biochem. Biophys. Res. Commun. 1991; 179: 10-16Crossref PubMed Scopus (20) Google Scholar, 19Okamoto T. Nishimoto I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8020-8023Crossref PubMed Scopus (51) Google Scholar) and Nishimoto et al. (20Nishimoto I. Ogata E. Okamoto T. J. Biol. Chem. 1991; 266: 12747-12751Abstract Full Text PDF PubMed Google Scholar) have suggested that the receptor binds to the guanyl nucleotide-binding protein, Gi-2, by means of a specific motif within the receptor's cytoplasmic domain. However, others have not been able to demonstrate interactions between G proteins and the receptor upon stimulation by IGF-II (21Braulke T. Korner C. Rosorius O. Nurnberg B. Baxter R.C. Gluckman P.D. Rosenfeld R.G. The Insulin-like Growth Factors and Their Regulatory Proteins. Elsevier Science, New York1994: 117-129Google Scholar, 22Korner C. Nurnberg B. Uhde M. Braulke T. J. Biol. Chem. 1995; 270: 287-295Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Indeed, many studies have not supported a model in which the CI-MPR acts as a signaling protein (23Braulke T. Tippmer S. Neher E. von Figura K. EMBO J. 1989; 8: 681-686Crossref PubMed Scopus (57) Google Scholar, 24Thakker J.K. DiMarchi R. MacDonald K. Caro J.F. J. Biol. Chem. 1989; 264: 7169-7175Abstract Full Text PDF PubMed Google Scholar, 25Guse A.H. Kiess W. Funk B. Kessler U. Berg I. Gercken G. Endocrinology. 1992; 130: 145-151Crossref PubMed Scopus (74) Google Scholar). Whereas the CI-MPR has been highly conserved in mammalian species, purification and characterization of the CI-MPR from the chicken and frog revealed that this receptor is unable to bind IGF-II (26Canfield W.M. Kornfeld S. J. Biol. Chem. 1989; 264: 7100-7103Abstract Full Text PDF PubMed Google Scholar, 27Clairmont K.B. Czech M.P. J. Biol. Chem. 1989; 264: 16390-16392Abstract Full Text PDF PubMed Google Scholar, 28Yang Y.W.-H. Robbins A.R. Nissley S.P. Rechler M.M. Endocrinology. 1991; 128: 1177-1189Crossref PubMed Scopus (41) Google Scholar). In addition, there is no evidence for an IGF-II-specific receptor in any other non-mammalian species examined thus far. This suggests that the CI-MPR acquired an IGF-II binding site after the separation of aves from mammals. Interestingly, the CI-MPR from a marsupial, the American opossum, does contain a binding site for IGF-II, albeit with an apparent 75-fold lower affinity for bovine IGF-II than the bovine receptor (1Dahms N.M. Brzycki-Wessell M.A. Ramanujam K.S. Seetharam B. Endocrinology. 1993; 133: 440-446Crossref PubMed Scopus (33) Google Scholar). It is not known whether the reported lower binding affinity is due to the use of a heterologous assay and possible amino acid differences between opossum and bovine IGF-II, or whether it indeed reflects the true binding affinity of this receptor. This information is important for determining where in evolution the CI-MPR acquired the ability to bind IGF-II. This knowledge, in turn, may lead to a greater understanding of the physiological role of this receptor in IGF-II action. To examine the marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (2Yandell C.A. Francis G.L. Wheldrake J.F. Upton Z. J. Endocrinol. 1998; 156: 195-204Crossref PubMed Scopus (8) Google Scholar), and we now report the purification and characterization of the kangaroo CI-MPR. The interaction of the kangaroo CI-MPR with IGF-II has been examined by radioreceptor assay, Western ligand blotting, and real-time biomolecular interaction analysis using both homologous and heterologous ligands. Furthermore, we have cloned the kangaroo CI-MPR cDNA sequence for the region proposed to be the IGF-II binding site on the mammalian receptor and have compared the sequences. Mannose-6-phosphate, aprotinin, vinyl sulfone, human γ-globulin, and 4-chloro-1-naphthol were purchased from Sigma/Aldrich. Sepharose 6B was purchased from Amersham Pharmacia Biotech, whereas the Hansenula holstii (NRRL Y-2154) phosphomannan was a generous gift from Dr. M. E. Slodki (United States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL). rhIGF-I and rhIGF-II were supplied by GroPep Pty Ltd. (Adelaide, Australia), and kIGF-II was purified as described previously (2Yandell C.A. Francis G.L. Wheldrake J.F. Upton Z. J. Endocrinol. 1998; 156: 195-204Crossref PubMed Scopus (8) Google Scholar). Kangaroo livers were obtained from wild western gray kangaroos (Macropus fuliginosus), which were professionally culled at Peterborough, South Australia, Australia, and immediately placed on solid CO2 until stored at −80 °C. Bovine livers from freshly slaughtered cattle were obtained from Agpro Abattoir (Gepps Cross, Australia) and snap-frozen in liquid nitrogen before being stored at −80 °C. Radioiodinated rhIGF-II and kIGF-II were prepared using chloramine T (29Francis G.L. Owens P.C. McNeil K.A. Wallace J.C. Ballard F.J. J. Endocrinol. 1989; 122: 681-687Crossref PubMed Scopus (39) Google Scholar) to specific activities of 230 × 103 and 8.8 × 103 Ci/mol, respectively. Antibodies directed against the rat IGF-II receptor (C6) were generously provided by Dr. C. L. Scott (Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia), and the goat anti-rabbit-horseradish peroxidase-conjugated antibody was purchased from DAKO (Botany, Australia). The lysosomal enzyme 4-sulfatase was recombinantly produced in Chinese hamster ovary cells (30Crawley A.C. Brooks D.A. Muller V.J. Peterson B.A. Isaac E.L. Bielicki J. King B.M. Boulter C.D. Moore A.J. Fazzalari N.L. Anson D.S. Byers S. Hopwood J.J. J. Clin. Invest. 1996; 97: 1864-1873Crossref PubMed Scopus (124) Google Scholar) and was generously provided by Prof. J. J. Hopwood (Department of Chemical Pathology, Women and Children's Hospital, Adelaide, Australia). 4-Sulfatase was iodinated using the lactoperoxidase method (31Tait J.F. Weinman S.A. Bradshaw R.A. J. Biol. Chem. 1981; 256: 11086-11092Abstract Full Text PDF PubMed Google Scholar) to a specific activity of 1.42 × 106 Ci/mol. The Bradford Protein Assay Reagent was purchased from Bio-Rad. Restriction enzymes (Nsi I and Eco RI), pGEM-7Zf vector, JM109 competent cells (subcloning efficiency), Wizard PCR DNA Purification System, Wizard Plus SV Miniprep Purification System, T4 DNA ligase, and M13 lacZ forward and reverse primers were purchased from Promega (Madison, WI). The RNeasy Mini Kit was purchased from Qiagen (Clifton Hill, Australia), whereas the ELONGASETMenzyme mix, 10 mm dNTP Mix, oligo(dT), and Superscript II RT were purchased from Life Technologies, Inc. The specific oligonucleotide primers as detailed below were synthesized by Life Technologies, Inc. The ExpandTM High Fidelity PCR System was obtained from Roche Molecular Biochemicals, and the ThermoSequenase sequencing kit was obtained from Amersham Pharmacia Biotech. H. holstii phosphomannan was hydrolyzed into core and small oligosaccharide fragments by mild acid treatment as described previously (32Bretthauer R.K. Kaczorowski G.J. Weise M.J. Biochemistry. 1973; 12: 1251-1256Crossref PubMed Scopus (62) Google Scholar). The phosphomannan core (1.4 g) was then coupled to vinyl sulfone-activated Sepharose 6B (50 ml) (33Hermanson G.T. Krishna-Mallia A. Smith P.K. Immobilized Affinity Ligand Techniques. Academic Press Inc., San Diego, CA1992Google Scholar), and the gel was transferred to a glass column 1 cm in diameter and 5 cm in length. The CI-MPR was isolated from kangaroo and bovine liver by phosphomannan-Sepharose affinity chromatography essentially as described by Dahms et al. (1Dahms N.M. Brzycki-Wessell M.A. Ramanujam K.S. Seetharam B. Endocrinology. 1993; 133: 440-446Crossref PubMed Scopus (33) Google Scholar), except that MnCl2 was omitted from the wash buffer (Buffer D: 50 mm Imidazole, 150 mm NaCl, 0.05% Triton X-100, 5 mm sodium glycerophosphate, pH 7.0) and the liver homogenate. The material from the phosphomannan affinity column was dialyzed against water and then lyophilized. The samples were concentrated to one-tenth of the original volume by resuspending the lyophilized material in 0.25 mm HEPES, pH 7.0. Purified receptor was concentrated as described above before electrophoresis. Samples and standards were boiled for 15 min in the presence of 2% SDS and applied to 6% or 8% SDS-polyacrylamide gels. Gels were run as described previously (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and either stained with Coomassie Blue or transferred to nitrocellulose membranes (35Hossenlopp P. Seurin D. Segovia-Quinson B. Hardouin S. Binoux M. Anal. Biochem. 1986; 154: 138-143Crossref PubMed Scopus (1199) Google Scholar). The membranes were probed with either 125I-labeled kIGF-II or125I-labeled rhIGF-II in the presence or absence of rhIGF-II (1 μg) as indicated in the figure legends. Membranes were also probed with I125-labeled 4-sulfatase in the presence or absence of 10 mm mannose 6-phosphate. Radiolabeled bands were visualized by Phosphor Imaging (ImageQuant, Molecular Dynamics). Immunoblotting was performed using a polyclonal anti-rat IGF-II receptor antibody (C6) (36Scott C.D. Baxter R.C. Endocrinology. 1987; 120: 1-9Crossref PubMed Scopus (43) Google Scholar) at a dilution of 1:3000. Immunoreactive protein bands were visualized by reaction with a 1:1000 dilution of goat anti-rabbit horseradish peroxidase-conjugated secondary antibody and 4-chloro-1-naphthol as detailed by the manufacturer. Binding of 125I-labeled rhIGF-II to the purified CI-MPR was determined as described by Scott and Baxter (36Scott C.D. Baxter R.C. Endocrinology. 1987; 120: 1-9Crossref PubMed Scopus (43) Google Scholar). All experiments were performed on a BIAcore 2000 system (Pharmacia Biosensor AB) using HBS buffer at 25 °C. Purified kIGF-II and rhIGF-II were coupled to the dextran-modified gold surface of a CM5 sensor chip by amine coupling as described in the BIAcore systems manual. Briefly, the dextran surface of the chip was activated with N-hydroxysuccinimide/N-ethyl-N′-(3-diethylaminopropyl) carbodiimide (40 μl) followed by the addition of a 3 mmIGF-II solution in 0.1 m CH3COONa, pH 4.6 (30 μl). The remaining activated groups were blocked with ethanolamine (40 μl). Surface densities of 390 and 620 resonance units were generated for hIGF-II and kIGF-II, respectively. Before data collection, several methods of surface regeneration after ligand binding were evaluated. It was found that washes with 1m NaCl/0.1 m HCl (30 μl) could remove the bound protein and also preserve the binding capacity of the biosensor surface. Before analysis, receptor preparations were dialyzed against HBS buffer, and protein concentrations were determined by the method of Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using Bio-Rad reagent and human γ-globulin as the standard. Different concentrations (20 μl) of either bovine or kangaroo CI-MPR were injected over the chip at a flow rate of 10 μl/min. A non-protein, blocked surface (flow cell 1) served as a blank, and sensorgrams from this flow cell were subtracted from all others. To investigate whether rebinding was apparent during the dissociation phase, HBS buffer containing a 10-fold excess of rhIGF-II (140 μl) was injected during the dissociation phase using the COINJECT command. Membrane and receptor concentrations were determined as outlined above. Human and kangaroo IGF-II concentrations were determined by reverse-phase high performance liquid chromatography as described previously (38Upton Z. Francis G.L. Kita K. Wallace J.C. Ballard F.J. J. Mol. Endocrinol. 1995; 14: 79-90Crossref PubMed Scopus (25) Google Scholar) using a wavelength of 214 nm and extinction coefficients of 31.701 and 31.69 g liter−1 cm−1, respectively. The cDNA sequences for the putative IGF-II binding region and the G protein recognition site were obtained using mRNA extracted from kangaroo liver, followed by reverse transcription-polymerase chain reaction (RT-PCR). Total kangaroo liver RNA was extracted using the RNeasy Mini Kit, and first-strand cDNA was synthesized using 0.5 μg of oligo(dT) primer and 200 units of Superscript II reverse transcriptase enzyme in a total volume of 20 μl (according to the manufacturer's protocols). Amplification of the putative IGF-II binding region was performed using two non-degenerate oligonucleotide primers that were based on the cDNA sequence of the human CI-MPR. The primer pair 5′-ATCAATGTCTGCAA-3′ (IGF-1) and 5′-CGTCCAGGAGAA-3′ (IGF-2) amplify a 732-base pair fragment spanning repeats 10 and 11. The PCR was carried out in a total volume of 20 μl containing 60 mm Tris-SO4 (pH 9.1), 18 mm (NH4)2SO4, 1.5 mm MgSO4, 0.2 mm dNTP, 0.5 unit of ExpandTM High Fidelity PCR enzyme mix (Roche Molecular Biochemicals), 100 ng of oligonucleotide primers, and 4.5 μl of cDNA template. After denaturation at 94 °C for 3 min, the PCR reaction proceeded for 40 cycles of 1 min at 94 °C (denaturation), 1 min at 50 °C (annealing), and 1 min at 72 °C (extension). After electrophoresis and ethidium bromide staining, the correct sized fragment was excised from the agarose gel, purified using Wizard PCR DNA Purification System, and subcloned into the vector (pGEM-7f). Amplification of the putative G protein recognition site was performed using two fully degenerate oligonucleotide primers deduced from the amino acid sequences from the chicken (39Zhou M. Ma Z. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9762-9766Crossref PubMed Scopus (66) Google Scholar), human (40Oshima 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), bovine (41Lobel P. Dahms N.M. Kornfeld S. J. Biol. Chem. 1988; 263: 2563-2570Abstract Full Text PDF PubMed Google Scholar), and mouse (42Szebenyi G. Rotwein P. Genomics. 1994; 19: 120-129Crossref PubMed Scopus (46) Google Scholar) CI-MPRs. This primer pair was based on the amino acid sequences of Gln2385-Glu2386-Asn2387-Glu2388-His2389(GP1) and Phe2474-His2475-Asp2476-Asp2477-Ser2478(GP2) (numbering is based on the human sequence). Amplification using primer set GP1 and GP2 generates a 315-base pair fragment. The PCR was carried out in a total volume of 50 μl containing 60 mmTris-SO4 (pH 9.1), 18 mm(NH4)2SO4, 1.5 mmMgSO4, 0.2 mm dNTP, 0.5 unit of ELONGASETM, 200 ng of oligonucleotide primers, and 2.5 μl of cDNA template. After denaturation at 94 °C for 3 min, the PCR reaction proceeded for 40 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 68 °C. The PCR product was purified by the Wizard PCR DNA Purification System and subcloned into the vector (pGEM-7f). Cloned PCR products were sequenced in both directions using a radiolabeled terminator cycle sequencing kit, ThermoSequenase (Amersham Pharmacia Biotech). To verify the sequence, two independent clones were sequenced. Phosphomannan-Sepharose was used to affinity purify the CI-MPR from membrane extracts of kangaroo and bovine livers. The Triton X-100 solubilized liver membranes were passed over the phosphomannan-Sepharose column, and the protein retained after washing with Buffer D was eluted with 4 mm mannose 6-phosphate. Peak fractions were pooled, concentrated, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig.1 a). The purified protein migrated as two forms, one with a molecular mass similar to that predicted for the CI-MPR, and a second approximately 50 kDa larger than expected. A faint band at approximately 60 kDa was also observed that most likely represents a small amount of cation-dependent mannose 6-phosphate receptor that was able to bind to the column in the absence of Mn2+. Incubation of the purified receptor under reducing conditions before SDS-PAGE inhibited the formation of the slower-migrating, high molecular mass protein, and only one band at approximately 250 kDa, along with the 60-kDa band, was present (data not shown). The purified protein was subjected to SDS-PAGE under nonreducing conditions, transferred to nitrocellulose membrane, and probed with a polyclonal antibody raised against the rat CI-MPR. Both the higher molecular mass proteins reacted with the anti-rat CI-MPR antibody (Fig. 1 b), whereas the smaller 60-kDa protein did not. The bovine and kangaroo CI-MPRs were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and probed with either 125I-labeled rhIGF-II or125I-labeled kIGF-II (Fig. 2, a and b). Both the kangaroo and bovine receptors specifically bound 125I-labeled IGF-II, and this binding could be displaced by the addition of excess unlabeled rhIGF-II (Fig.2, a and b), but not IGF-I (data not shown). However, the kangaroo CI-MPR bound IGF-II with an apparent lower affinity than the bovine CI-MPR. Moreover, the low affinity of the kangaroo receptor for IGF-II did not appear to be dependent upon whether the heterologous or homologous ligand was used as the probe. On the other hand, when probed with 125I-labeled 4-sulfatase, both the kangaroo and bovine CI-MPRs bound this lysosomal enzyme with equal apparent affinities (Fig. 2 c). Again, this binding was specific because the radioligand was displaced by the addition of 10 mm mannose 6-phosphate. Both species of the receptor (i.e. both bands above 220 kDa) were able to bind IGF-II and 4-sulfatase. Radioreceptor binding assays confirmed the lower binding affinity of the kangaroo CI-MPR for IGF-II. The specific binding of hIGF-II to the kangaroo and bovine CI-MPRs was proportional to the amount of receptor added (Fig.3, a and b). However, greater amounts of kangaroo receptor than bovine receptor were required to achieve a similar level of specific binding. For example, at the highest concentrations tested, 34 and 2460 ng for the bovine and kangaroo CI-MPRs, respectively, specific binding was determined to be 52% and 34% of the radioligand added. Similarly, in an assay measuring the ability of unlabeled rhIGF-II to compete with125I-labeled rhIGF-II for binding to the CI-MPR, specific binding of radiolabeled rhIGF-II to the kangaroo receptor (100 ng/tube) in the absence of competing unlabeled hIGF-II was only 5% of the total counts added (Fig. 4). However, specific binding of the bovine receptor (20 ng/tube) in the absence of added unlabeled rhIGF-II was approximately 80% of the total counts added (Fig. 4), with half-maximal inhibition of radiolabeled rhIGF-II binding to the bovine receptor observed with the addition of 0.6 nmrhIGF-II. Thus, this assay was not useful for analysis of the relative binding affinities of these receptors because of the large discrepancy in the amounts of protein required to obtain a competitive binding curve. Additionally, competitive binding curves were not performed using 125I-labeled kIGF-II because of the low specific activity of this radioligand. Characterization of the interaction between IGF-II and the kangaroo CI-MPR was therefore further investigated by real-time biomolecular interaction analysis.Figure 4Competitive binding curve for the binding of125I-labeled rhIGF-II to the purified bovine (•) and kangaroo (▪) CI-MPR. Increasing amounts of unlabeled rhIGF-II were added to 20 ng of bovine or 100 ng of kangaroo CI-MPR and 10,000 cpm of 125I-labeled rhIGF-II. Nonspecific binding as determined in the absence of receptor was less than 10% of the total counts added and has been subtracted. Specific binding is expressed as a percentage of total counts added, and values represent the mean ± S.D. for one representative experiment performed in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Preliminary experiments examining the binding of kangaroo CI-MPR to IGF-II using real-time biomolecular interaction indicated that the increase in resonance signals when analyzing different concentrations of receptor was dose-dependent (data not shown). However, the dissociation of both the bovine and kangaroo receptors appeared to be very slow. Therefore, we examined the possibility that the receptors were rebinding during the dissociation phase, thus giving a misleading indication of the dissociation rate. Co-injection of excess rhIGF-II immediately after injection of the receptor indicated that significant rebinding was occurring, and all subsequent analysis was performed using a co-injection of excess rhIGF-II. Analysis of the sensorgrams indicated that whereas the association rates of the bovine and kangaroo CI-MPRs are similar, the lower affinity of the kangaroo CI-MPR is due to the fast dissociation of this receptor from IGF-II (Fig.5). Furthermore, the dissociation of the kangaroo CI-MPR appears to be faster when binding to hIGF-II than kIGF-II, whereas the dissociation rate of the bovine CI-MPR is very similar for both IGF-II proteins. A primer pair designed to span the proposed IGF-II binding region was used for the RT-PCR amplification of mRNA isolated from kangaroo liver. A product of approximately 750 base pairs was isolated, cloned, and sequenced (Fig.6 a). The deduced amino acid sequence of the proposed IGF-II binding site, residues 1508–1575, revealed that there is a 60% identity between the kangaroo and bovine CI-MPRs (41Lobel P. Dahms N.M. Kornfeld S. J. Biol. Chem. 1988; 263: 2563-2570Abstract Full Text PDF PubMed Google Scholar) and a 49% identity between the kangaroo and chicken receptors (39Zhou M. Ma Z. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9762-9766Crossref PubMed Scopus (66) Google Scholar) (Fig. 6 b). Of the 27 amino acids that differ between the bovine and kangaroo receptors, 22 are found in the N-terminal region of repeat 11 (1533–1575). The cDNA sequence of the putative G protein recognition site was obtained using RT-PCR, and a PCR product of approximately 500 base pairs was detected. Analysis of the amino acid sequence deduced from the cDNA sequence and comparison with the sequences reported for bovine (41Lobel P. Dahms N.M. Kornfeld S. J. Biol. Chem. 1988; 263: 2563-2570Abstract Full Text PDF PubMed Google Scholar), chicken (39Zhou M. Ma Z. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9762-9766Crossref PubMed Scopus (66) Google Scholar), and human CI-MPRs (40Oshima 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) revealed interesting differences between the species (Fig. 7). Seven of the 14 amino acids comprising the putative G protein binding site differ between the kangaroo and bovine CI-MPRs, whereas 6 of the 14 differ between the kangaroo and chicken sequences. However, 4 of the 14 differ between kangaroo and human CI-MPRs, 3 of the 14 differ between human and bovine CI-MPRs, and 1 of the 14 differs between mouse and human receptors. The CI-MPR from the opossum has previously been shown to bind bovine IGF-II with a 75-fold lower apparent affinity than the bovine receptor (1Dah" @default.
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