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J. Biol. Chem., Vol. 280, Issue 22, 20932-20936, June 3, 2005

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Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor^*

Jonathan Whittaker¶ and Linda Whittaker

From the Departments of Nutrition and Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4906

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1–470) and at the C terminus of the subunit (amino acids 705–715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the K_d of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg¹⁴, Phe⁶⁴, Phe⁷⁰⁵, Glu⁷⁰⁶, Tyr⁷⁰⁸, Asn⁷¹¹, and Val⁷¹⁵ inactivated the receptor. Alanine mutation of Asn¹⁵ resulted in a 20-fold decrease in affinity, whereas mutations of Asp¹², Gln³⁴, Leu³⁶, Leu³⁷, Leu⁸⁷, Phe⁸⁹, Tyr⁹¹, Lys¹²¹, Leu⁷⁰⁹, and Phe⁷¹⁴ all resulted in 4–10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn¹⁵, Leu³⁷, Asp⁷⁰⁷, Leu⁷⁰⁹, Tyr⁷⁰⁸, Asn⁷¹¹, Phe⁷¹⁴, and Val⁷¹⁵, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn¹⁵, Gln³⁴, and Phe⁸⁹ on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC₅₀ commensurate with their effect on the affinity of the receptor for insulin.

Received for publication, October 4, 2004 , and in revised form, March 11, 2005.

^* This work was supported by National Institutes of Health Grant 5 R01 DK065890 and Juvenile Diabetes Research Foundation International Grant 1-2000-198 (to J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Nutrition, Case Western Reserve University, Dental Bldg., 2123 Abington Rd., Rm. 201, Cleveland, OH 44106-4906. Tel.: 216-368-6625; Fax: 216-368-6644; E-mail: Jonathan.Whittaker{at}case.edu.

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