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An Insulin Receptor Mutant (Asp707→ Ala), Involved in Leprechaunism, Is Processed and Transported to the Cell Surface but Unable to Bind Insulin

1996; Elsevier BV; Volume: 271; Issue: 31 Linguagem: Inglês

10.1074/jbc.271.31.18719

1083-351X

Leen M. ‘t Hart, Dick Lindhout, Gerard C.M. van der Zon, Hülya Kayserilli, Memnune Yüksel Apak, Wim J. Kleijer, Eric R. van der Vorm, J. A. Maassen,

Metabolism, Diabetes, and Cancer

We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) α subunit from a leprechaun patient, changing Asp707 into Ala. Fibroblasts from this patient had no high affinity insulin binding sites. To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells. Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to α and β subunits. No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of 125I-labeled insulin by disuccinimidyl suberate to these cells failed to label the mutant α subunit. Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR α and β subunits, showing its presence on the cell surface. On solubilization of the mutant insulin receptor in Triton X-100-containing buffers, 125I-insulin was efficiently cross-linked to the receptor α subunit by disuccinimidyl suberate.These studies demonstrate that Ala707 IR is normally processed and transported to the cell surface and that the mutation distorts the insulin binding site. Detergent restores this site. This is an example of a naturally occurring mutation in the insulin receptor that affects insulin binding without affecting receptor transport and processing. This mutation points to a major contribution of the α subunit carboxyl terminus to insulin binding. We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) α subunit from a leprechaun patient, changing Asp707 into Ala. Fibroblasts from this patient had no high affinity insulin binding sites. To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells. Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to α and β subunits. No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of 125I-labeled insulin by disuccinimidyl suberate to these cells failed to label the mutant α subunit. Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR α and β subunits, showing its presence on the cell surface. On solubilization of the mutant insulin receptor in Triton X-100-containing buffers, 125I-insulin was efficiently cross-linked to the receptor α subunit by disuccinimidyl suberate. These studies demonstrate that Ala707 IR is normally processed and transported to the cell surface and that the mutation distorts the insulin binding site. Detergent restores this site. This is an example of a naturally occurring mutation in the insulin receptor that affects insulin binding without affecting receptor transport and processing. This mutation points to a major contribution of the α subunit carboxyl terminus to insulin binding. INTRODUCTIONInsulin induces mitogenic and metabolic responses in cells. In addition, in muscle and adipose tissues, glucose transporters become translocated to the plasma membrane (1Garvey W.T. Birnbaum M.J. Ferrannini E. Bailliere's Clinical Endocrinology and Metabolism. Balliere Bindall, London1995: 785Google Scholar). These responses require tyrosine kinase activity in the cytoplasmic tail of the insulin receptor, which is activated on binding of insulin (2Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.C. Tsubokowa M. Mason A. Seeburg P.H. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1505) Google Scholar, 3Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (964) Google Scholar). The insulin receptor is synthesized as a proreceptor, which after proteolytic cleavage into α and β subunits and extensive glycosylation in the Golgi apparatus, appears predominantly as an α2β2 tetramer on the cell surface (4Lane M.D. Ronnett G. Slieker L.J. Kohanski R.A. Olson T.L. Biochimie (Paris). 1985; 67: 1069-1080Crossref PubMed Scopus (19) Google Scholar).A number of naturally occurring mutations in the insulin receptor have been found to associate with diseases of severe insulin resistance (5Taylor S.I. Cama A. Accili A. Barbetti F. Quon M.J. Luz Sierra M. Suzuki Y. Koller E. Levy-Toledano R. Wertheimer E. Moncada V.Y. Kadowaki H. Kadowaki T. Endocr. Rev. 1992; 13: 566-595Crossref PubMed Scopus (270) Google Scholar). Missense mutations in the cytoplasmic domain of the receptor are often seen in patients with type A insulin resistance. These mutant receptors are processed to α2β2 tetramers and transported to the cell surface. Usually they retain their insulin binding properties. The impaired activation of the receptor tyrosine kinase contributes to the development of the syndrome of insulin resistance. Missense mutations in the homozygous or compound heterozygous state in the extracellular part of the receptor are often associated with syndromes of extreme insulin resistance such as leprechaunism (MIM 246200) or Rabson Mendenhall syndrome (MIM 262190) (5Taylor S.I. Cama A. Accili A. Barbetti F. Quon M.J. Luz Sierra M. Suzuki Y. Koller E. Levy-Toledano R. Wertheimer E. Moncada V.Y. Kadowaki H. Kadowaki T. Endocr. Rev. 1992; 13: 566-595Crossref PubMed Scopus (270) Google Scholar, 6McKusick V.A. McKusick V.A. Mendelian Inheritance in Man. 11th Ed. The Johns Hopkins University Press, Baltimore1994Google Scholar). Many of the missense mutations involved lead to a loss of high affinity insulin binding sites on cells. These mutations are predicted to affect the folding of the α subunit and the formation of correct disulfide bonds. As a result, the proreceptor is retained in the endoplasmic reticulum, and no receptors are transported to the cell surface (7Van der Vorm E.R. Van der Zon G.C.M. Möller W. Krans H.M.J. Lindhout D. Maassen J.A. J. Biol. Chem. 1992; 267: 66-71Abstract Full Text PDF PubMed Google Scholar, 8Kadowaki T. Kadowaki H. Accili D. Yazaki Y. Taylor S.I. J. Biol. Chem. 1991; 266: 21224-21231Abstract Full Text PDF PubMed Google Scholar). Another situation that leads to a decreased number of functional receptors on the cell surface is represented by the naturally occurring mutation Glu460. This receptor undergoes enhanced internalization and degradation on binding of insulin (9Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar).We have recently encountered leprechaun patient HO. This patient was found to have a mutation in the insulin receptor, which interferes with insulin binding, probably by a direct effect on the structure of the insulin binding site.DISCUSSIONLeprechaun patient HO is homozygous for a mutation in the insulin receptor, which replaces Asp707 by Ala. This mutant receptor is normally processed into α and β subunits and transported to the cell surface. In the α chain, the proper positioning of Cys residues is essential for correct disulfide bond formation and for the formation of functional insulin receptors, which can be transported to the cell surface. A number of mutations in the α chain, found in patients with leprechaunism or Rabson Mendenhall syndrome, lead to transport-defective receptors, which are retained in the endoplasmic reticulum. Those mutations often involve amino acid residues near Cys residues, and the nature of the amino acid substitution is such that it is likely that they affect the folding of the polypeptide chain (5Taylor S.I. Cama A. Accili A. Barbetti F. Quon M.J. Luz Sierra M. Suzuki Y. Koller E. Levy-Toledano R. Wertheimer E. Moncada V.Y. Kadowaki H. Kadowaki T. Endocr. Rev. 1992; 13: 566-595Crossref PubMed Scopus (270) Google Scholar). Residue 707 is located near the carboxyl terminus of the α chain. This region has a high probability of existing in an α helical conformation. No Cys residues involved in disulfide bond formation are found in this part of the receptor. Because Ala707 IR is transported to the cell surface, it is expected that the mutation has no major effect on the overall folding of the α subunit. This situation is corroborated by protein structure predictions, which indicate that substituting Ala for Asp has no major effect on the probability of the carboxyl terminus adopting an α helical configuration. Because of the smaller side chain of Ala compared with Asp, the helix may become more flexible at that site.Despite the predicted absence of a major effect on protein folding, Ala707 IR is unable to bind insulin when expressed on cells. The absence of insulin binding to cells expressing the mutant insulin receptor is not the result of enhanced internalization and receptor degradation, a situation seen in case of the Glu460 mutant (9Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar). We conclude that the loss of insulin binding to cells expressing Ala707 IR is due to a direct effect of the mutation on the process of insulin binding. This loss of binding may, in principle, result from an essential contribution of Asp707 to the interaction of the receptor with insulin or to a conformational change induced by Ala707, which affects the structure of the insulin binding pocket.When insulin binding to Ala707 IR is determined in the presence of Triton X-100, binding of insulin to the mutant receptor is seen, and this binding induces receptor autophosphorylation. The dose-response relationship for insulin-induced receptor autophosphorylation shows an unchanged ED50 and a slightly, but significantly, lower autophosphorylation at low insulin concentrations. These observations indicate that in the presence of detergent, the mutation has a minor effect on the kinetics of insulin binding and that Asp707 does not provide interactions that are essential for insulin binding. Rather, the data suggest that the introduction of Ala707 induces a change in the positioning of the carboxyl-terminal α helix, which prevents insulin from binding when the receptor is on the cell surface. Detergent somehow is able to restore insulin binding. It may do so by making the structure of the carboxyl-terminal part of the receptor α subunit more flexible, thereby allowing the carboxyl terminus of Ala707 IR to adopt a position that allows insulin to bind. Another possibility is that the mutant receptor has gained the ability to bind to another protein, which prevents insulin from binding. We think that this situation is unlikely, because we do not observe coprecipitating proteins in insulin receptor immune precipitates from metabolically labeled Ala707 IR-expressing cells that are absent in cells expressing WT IRs.Previously, sites on the insulin receptor that are important for the binding of insulin were identified in the NH2-terminal region, especially Phe89 (13Yip C.C. J. Cell. Biochem. 1992; 48: 19-25Crossref PubMed Scopus (31) Google Scholar, 14Andersen A.S. Kjeldsen T. Wiberg F.C. Vissing H. Schaffer L. Rasmussen J.S. DeMeyts P. Moller N.P.H. J. Biol. Chem. 1992; 267: 13681-13686Abstract Full Text PDF PubMed Google Scholar, 15DeMeyts P. Gu J.-L. Shymko R.M. Kaplan B.E. Bell G.I. Whittaker J. Mol. Endocrinol. 1990; 4: 409-416Crossref PubMed Scopus (70) Google Scholar), the cysteine-rich region (16Yip C.C. Hsu H. Patel R.G. Hawley D.M. Maddux B.A. Goldfine I.D. Biochem. Biophys. Res. Commun. 1988; 157: 321-329Crossref PubMed Scopus (61) Google Scholar, 17Rafaeloff R. Patel R. Yip C. Goldfine I.D. Hawley D.M. J. Biol. Chem. 1989; 264: 15900-15904Abstract Full Text PDF PubMed Google Scholar, 18Gustafson T.A. Rutter W.J. J. Biol. Chem. 1990; 265: 18663-18667Abstract Full Text PDF PubMed Google Scholar), and the region encoded by exons 6 and 7 (9Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar, 19Fabry M. Schaefer E. Ellis L. Kojro E. Fahrenholz F. Brandenburg D. J. Biol. Chem. 1992; 267: 8950-8956Abstract Full Text PDF PubMed Google Scholar, 20Zhang B. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9858-9862Crossref PubMed Scopus (78) Google Scholar). Also, an important contribution of Ser323 to the formation of a high affinity insulin binding site on the receptor has been detected by the study of an insulin-resistant patient. Substituting Leu for Ser323 leads to a severe impairment in insulin binding without significantly altering the processing or cell surface expression of the receptor (21Taouis M. Levy-Toledano R. Roach P. Taylor S.I. Gorden P. J. Biol Chem. 1994; 269: 14912-14918Abstract Full Text PDF PubMed Google Scholar).Subsequently, another site on the receptor has been identified that may contribute to insulin binding. Ultraviolet light-induced cross-linking of B25 azidophenylalanine insuline was found to occur in a peptide comprising residues 704-718 (22Kurose T. Pashmforoush M. Yoshimasa Y. Carroll R. Schwarz G.P. Burke G.T. Katsoyannis P.G. Steiner D.F. J. Biol. Chem. 1994; 269: 29190-29197Abstract Full Text PDF PubMed Google Scholar). This finding suggests a direct interaction between insulin and the carboxyl terminus of the receptor. Also in agreement with a role of the carboxyl terminus in insulin binding is the observation that a secreted form of the extracellular part of the insulin receptor, in which the carboxyl terminus of the α subunit is lacking, has a low affinity for insulin (23Schaefer E.M. Siddle K. Ellis L. J. Biol. Chem. 1990; 265: 13248-13253Abstract Full Text PDF PubMed Google Scholar).The region comprising amino acids 700-718 of the insulin receptor has a high probability to adapt an α helical conformation. As we see a complete loss of insulin binding to the mutant receptor in the absence of detergent, whereas addition of detergent restores binding, it is likely that the precise positioning of the carboxyl terminus is essential to enable binding of insulin. In case of Ala707 IR, this positioning may be somewhat changed, either due to an increased flexibility because of an Ala residue at 707, or because Asp stabilizes the spatial positioning of the α helix in a conformation that allows binding of insulin. Somehow, detergent is able to restore the correct spatial positioning of the carboxyl terminus of the α chain, thereby restoring the insulin binding site and insulin-induced receptor autophosphorylation. Although the precise mechanism by which Ala707 induces a complete loss of insulin binding is not known, our data indicate that the carboxyl terminus of the α subunit provides an essential contribution to the process of insulin binding. Besides, our results show that solubilization by detergent can markedly affect the interpretation of data on mutated receptors when insulin binding characteristics are considered.Other evidence suggesting a contribution of amino acid residues in the carboxyl terminus of the α subunit to the binding of insulin comes from insulin binding studies of proreceptors. When mutations are present in the tetrabasic processing site at the junction of α and β subunits, proreceptors are generated, which appear on the cell surface (24Yoshimasa Y. Seino S. Whittaker J. Kakehi T. Kosaki A. Kuzuya H. Imura H. Bell G.I. Steiner D.F. Science. 1988; 240: 784-787Crossref PubMed Scopus (195) Google Scholar). It was found that proreceptors without exon 11 have markedly reduced insulin binding compared with proreceptors with exon 11, which bind insulin with near normal affinity. Cleavage of proreceptors without exon 11 into α and β subunits markedly restored insulin binding. These findings suggest that the presence or absence of 12 amino acids encoded by exon 11 affects the folding and/or conformation of the α subunit carboxyl-terminal region in the proreceptor to confer altered binding of insulin. In the absence of exon 11, this region is in a strained conformation that disrupts the insulin binding site. Proteolytic cleavage at the α-β junction releases this constraint (25Pashmforoush M. Yoshimasa Y. Steiner D.F. J. Biol. Chem. 1994; 269: 32639-32648Abstract Full Text PDF PubMed Google Scholar).After completion of the manuscript, a publication appeared describing alanine-scanning mutagenesis of carboxyl-terminal amino acids in the insulin receptor α subunit (26Mynarcik D.C. Yu G.Q. Whittaker J. J. Biol. Chem. 1996; 271: 2439-2442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This study included the replacement of Asp707 by Ala. The results show that when a secreted form of the α subunit is examined for insulin binding, replacement of Asp707 by Ala did not affect insulin binding properties. Replacements of several other amino acid residues in the carboxyl-terminal region by Ala did affect insulin binding. These results support our conclusion that Asp707 as such is not essential for insulin binding when receptors are in the solubilized state. Rather, when the insulin receptor is present on the cell surface, Asp707 is essential in keeping the carboxyl-terminal tail in a spatial conformation, which allows insulin to bind. Ala707 IR represents the first naturally occurring missense mutation in the carboxyl-terminal part of the insulin receptor α subunit that affects binding of insulin in the absence of an effect on receptor processing or transport. INTRODUCTIONInsulin induces mitogenic and metabolic responses in cells. In addition, in muscle and adipose tissues, glucose transporters become translocated to the plasma membrane (1Garvey W.T. Birnbaum M.J. Ferrannini E. Bailliere's Clinical Endocrinology and Metabolism. Balliere Bindall, London1995: 785Google Scholar). These responses require tyrosine kinase activity in the cytoplasmic tail of the insulin receptor, which is activated on binding of insulin (2Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.C. Tsubokowa M. Mason A. Seeburg P.H. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1505) Google Scholar, 3Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (964) Google Scholar). The insulin receptor is synthesized as a proreceptor, which after proteolytic cleavage into α and β subunits and extensive glycosylation in the Golgi apparatus, appears predominantly as an α2β2 tetramer on the cell surface (4Lane M.D. Ronnett G. Slieker L.J. Kohanski R.A. Olson T.L. Biochimie (Paris). 1985; 67: 1069-1080Crossref PubMed Scopus (19) Google Scholar).A number of naturally occurring mutations in the insulin receptor have been found to associate with diseases of severe insulin resistance (5Taylor S.I. Cama A. Accili A. Barbetti F. Quon M.J. Luz Sierra M. Suzuki Y. Koller E. Levy-Toledano R. Wertheimer E. Moncada V.Y. Kadowaki H. Kadowaki T. Endocr. Rev. 1992; 13: 566-595Crossref PubMed Scopus (270) Google Scholar). Missense mutations in the cytoplasmic domain of the receptor are often seen in patients with type A insulin resistance. These mutant receptors are processed to α2β2 tetramers and transported to the cell surface. Usually they retain their insulin binding properties. The impaired activation of the receptor tyrosine kinase contributes to the development of the syndrome of insulin resistance. Missense mutations in the homozygous or compound heterozygous state in the extracellular part of the receptor are often associated with syndromes of extreme insulin resistance such as leprechaunism (MIM 246200) or Rabson Mendenhall syndrome (MIM 262190) (5Taylor S.I. Cama A. Accili A. Barbetti F. Quon M.J. Luz Sierra M. Suzuki Y. Koller E. Levy-Toledano R. Wertheimer E. Moncada V.Y. Kadowaki H. Kadowaki T. Endocr. Rev. 1992; 13: 566-595Crossref PubMed Scopus (270) Google Scholar, 6McKusick V.A. McKusick V.A. Mendelian Inheritance in Man. 11th Ed. The Johns Hopkins University Press, Baltimore1994Google Scholar). Many of the missense mutations involved lead to a loss of high affinity insulin binding sites on cells. These mutations are predicted to affect the folding of the α subunit and the formation of correct disulfide bonds. As a result, the proreceptor is retained in the endoplasmic reticulum, and no receptors are transported to the cell surface (7Van der Vorm E.R. Van der Zon G.C.M. Möller W. Krans H.M.J. Lindhout D. Maassen J.A. J. Biol. Chem. 1992; 267: 66-71Abstract Full Text PDF PubMed Google Scholar, 8Kadowaki T. Kadowaki H. Accili D. Yazaki Y. Taylor S.I. J. Biol. Chem. 1991; 266: 21224-21231Abstract Full Text PDF PubMed Google Scholar). Another situation that leads to a decreased number of functional receptors on the cell surface is represented by the naturally occurring mutation Glu460. This receptor undergoes enhanced internalization and degradation on binding of insulin (9Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar).We have recently encountered leprechaun patient HO. This patient was found to have a mutation in the insulin receptor, which interferes with insulin binding, probably by a direct effect on the structure of the insulin binding site.