Abstract
Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.
Highlights
Evolutionary history [1,2,3]
We have explored the possibility that sequence differences in the A- and B-domains between insulin and the insulin-like growth factors (IGFs) are involved in the lower affinity of the IGFs for the insulin receptor (IR)-A and whether they contribute to the higher affinity of IGF-II than IGF-I for the IR-A
By analyzing numerous single substituted insulin analogues, single substituted IGF-I analogues and multiple substituted insulin and IGF analogues, we clearly show that a small number of amino acid differences in the A- and B-chain of insulin and the IGFs are sufficient to explain why the IGFs bind with lower affinity than insulin for the IR-A
Summary
C-domain and, to a lesser extent, the D-domain contribute to the higher affinity of IGF-II than IGF-I for the IR-A and suggested that either domain length or charge differences could be responsible. We have explored the possibility that sequence differences in the A- and B-domains between insulin and the IGFs are involved in the lower affinity of the IGFs for the IR-A and whether they contribute to the higher affinity of IGF-II than IGF-I for the IR-A. By analyzing numerous single substituted insulin analogues, single substituted IGF-I analogues and multiple substituted insulin and IGF analogues, we clearly show that a small number of amino acid differences in the A- and B-chain of insulin and the IGFs are sufficient to explain why the IGFs bind with lower affinity than insulin for the IR-A. Our results show that there is room for the C-domain of IGF-I and IGF-II in the binding pocket of the IR-A
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