To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor a-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. We have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain ofhigh affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.The polypeptide hormone insulin plays an essential role in metabolic regulation (1-3). It exerts its physiological effects through its interaction with a specific high-affinity receptor, which is an integral cell surface membrane glycoprotein with a relative molecular mass of about 400 kDa (4-6). The receptor is a disulfide-linked heterotetramer, made up of two a-and two B-subunits (7-12). Its ligand binding domains are formed by the extracellular a-subunits, whereas the /3-subunits consist of a short extracellular domain, a single transmembrane segment, and an intrinsic intracellular tyrosine protein kinase domain involved in signal transduction (13-15).The insulin-like growth factor I (IGF-I) receptor shows extensive similarity to the insulin receptor in amino acid sequence, domain structure, and signaling mechanism (11,16). Although insulin and IGF-I are very similar in amino acid sequence, they bind only weakly to the receptor for the other hormone (17,18).Ligand binding affinity and specificity are central to receptor activation, regulation, and function. Chimeric molecules comprising parts of related receptors have been useful for elucidating relationships between structure and specificity in several systems (e.g., refs. 19-21). In a previous study we showed that it is possible to convert the insulin receptor into a receptor with high affinity for IGF-I and low affinity for insulin by substituting an insulin receptor cDNA fragment (exons 1, 2, and 3) with that for the corresponding . This suggested to us that the ligand specificity of both receptors resides in the same region. Here, we extend this approach to a more detailed analysis of the ligand specificity of this receptor family. The first 68 N-terminal amino acids of the insulin receptor are found to determine insulin specificity. The corresponding 62 N-t...
Phenolic additives widely used for the preservation of insulin preparations can have a profound effect on the hormone's conformation in solution. m-Cresol, for instance, increases the circular dichroism in the far ultraviolet by 10-20%, corresponding to an increase in helix, and around 255 nm. The CD-spectral changes are strikingly similar to those brought about by halide ions which have been identified to reflect the 2Zn-*4Zn insulin transition. Its most prominent element is the helix formation at the B-chain N-terminus. In both cases the changes fail to occur with dimeric insulin in the absence of 2 and with monomeric des-(B26-B30)-insulin. In the presence of Ni 20 which is unable to replace 2 in 4Zn insulin for coordinative reasons, the effect of m-cresol is impeded. m-Cresol thus induces a transition identical with or closely similar to the 2Zn~*4Zn transformation. 2Zn insulin crystals, when soaked in m-cresol containing solvents, are destroyed. Crystals grown in the presence of m-cresol, however, are monoclinic and containing symmetrical hexamers of, notably, 4Zn conformation. Phenol, o-and p-cresol, w-nitrophenol, Nipagin and benzene were further additives tested, all of them inducing largely the same spectral effects except for benzene. The results presented corroborate the close correspondence of insulin's structure in solution and in the crystal as well as insulin's capacity for structural variation. Phenol-induzierter Strukturübergang von Insulin in LösungZusammenfassung: Phenolische Konservierungsmittel für Insulinpräparate können die Konformation des Hormons in Lösung stark beeinflussen. /77-Kresol z.B. steigert den Circulardichroismus im fernen UV um 10 bis 20% im Sinne einer Erhöhung des Helixgehaltes sowie auch im Bereich um 255 nm. Die CD-spektralen Effekte entsprechen genau den durch Halidanionen herbeigeführten, die als Ausdruck des 2Zn-»4Zn-Überganges identifiziert worden sind. Dessen einprägsamstes Merkmal ist die Überführung der N-terminalen B-Kette in eine Helix. In beiden Fällen bleiben die Effekte mit dimerem Insulin in Abwesenheit von Zn 20 sowie mit monomerem Des-(B26-B30)-insulin aus. In Gegenwart von Ni 2 ®, das Zn 2 ® in 4Zn-Insulin aus koordinativen Gründen nicht ersetzen kann, zeigen sich die Effekte nur ansatzweise. m-Kresol bewirkt folglich eine dem 2Zn-*4Zn-Übergang entsprechende Konformationsänderung des Insulins in Lösung.
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