N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr7 and Leu8 Important for Binding to Insulin and IGF-I Receptors and Leu8 Critical for All IGF-II Functions

Abstract

To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The binding affinity and mitogenic activity of these rIGF-II mutants were the affinity of and for insulin and of of the affinity of to and of of the to the the affinity of deletion for these to of of Gly8 for Leu in rIGF-II the affinity of mutant for the and insulin to the the of and role in of the in the affinity for binding of the mutants to these the in mitogenic for the affinity of for of of the of des-(1-5)-, and for these of and for binding to insulin and and for of To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The binding affinity and mitogenic activity of these rIGF-II mutants were the affinity of and for insulin and of of the affinity of to and of of the to the the affinity of deletion for these to of of Gly8 for Leu in rIGF-II the affinity of mutant for the and insulin to the the of and role in of the in the affinity for binding of the mutants to these the in mitogenic for the affinity of for of of the of des-(1-5)-, and for these of and for binding to insulin and and for of growth growth insulin-like growth growth insulin-like growth and of and and the of the of and and the The and of and by and the and binding of the and affinity to receptors, and IGF-II, and to affinity to the and insulin of its affinity binding to the for binding to the and site-directed and in the and were for binding to the insulin and receptors, and in the were for its binding to these in and in the in and were for the binding of to of the N-terminal of for of mitogenic activity in and and The in mitogenic to the of in the affinity of for the growth to To the of of deletion mutants of the N-terminal of rIGF-II prepared by and affinity for affinity for the the affinity of for IGF and of of and the affinity of mutants for IGF and insulin of of The and The of rIGF-II by the N-terminal region the prepared of deletion mutants and substitution mutant in the N-terminal to define the region of the to the binding of rIGF-II to and and were and rIGF-II were the of rIGF-II to the affinity of binding of to prepared by the insulin and were were of the and were to The The to the prepared The by affinity and to to insulin and affinity The of the by by were by and by the of rIGF-II for in Escherichia in the of The N-terminal substitution deletion mutants of rIGF-II were by site-directed the in The substitution mutant prepared the of the mutant by its affinity to the insulin the of the N-terminal deletion des-(1-5)-, des-(1-7)-, and N-terminal by and of mutants were expressed in of these were by The mutant were and the were by of the rIGF-II mutants for by and and N-terminal of the in the the by to were by the were for rIGF-II and of in and and in the were to of and were and of were to the of of rIGF-II to and for the binding of and to insulin and by and rIGF-II mutants were of of were in of of and of were by the of of and of were and in of the mutants for were by prepared of were in of of of and of The of the mutants for were were in of of of and of were by the were and to were the the of in the of of and the of in the of The for the binding of these to the were the of of of the of rIGF-II of rIGF-II mutants were by the of of were in were in of rIGF-II rIGF-II mutants in were to the in for the of the were for of and by The of by of in by rIGF-II and the rIGF-II and The expressed the of of rIGF-II N-terminal deletion des-(1-5)-, des-(1-7)-, and and the substitution mutant were prepared and by and The of the and and and and in the deletion mutants were by and the mutant to The N-terminal for des-(1-5)-, des-(1-7)-, and were by to and The of the and of these mutants in of des-(1-7)-, and mutants were of the rIGF-II in The were of the mutants were of the of and the the rIGF-II The by and the of the of rIGF-II and rIGF-II and of of rIGF-II rIGF-II and were in of rIGF-II for to and for the binding of insulin and rIGF-II to insulin were and The for the binding of and rIGF-II to were and to in for these The of for rIGF-II binding to the in in the the of to of the in to the of affinity of binding of mutant to the in and of to of the binding of rIGF-II mutants to and of binding to insulin and by insulin rIGF-II and The the of to and the of in the of of and the of in its in The deletion of the and the N-terminal of the the of in the affinity of the rIGF-II mutants for the insulin and and to the insulin and and of the affinity of rIGF-II the to the the binding of the mutants to the the of des-(1-5)-, and for the were and of of to the of N-terminal of its binding to IGF the for the binding of rIGF-II to of the in the to the in the The of and for the were and of of rIGF-II The affinity of the for the for the for the affinity of the for of the of binding of the mutants to and deletion of the N-terminal the affinity of the mutants for the the binding of the rIGF-II to for the N-terminal to The binding of and of of these to for The of its to to the the binding affinity of the mutant by to of the the and of by and of the of rIGF-II the of in to the and for the binding of to were for the of the the of of of the N-terminal deletion mutants of rIGF-II the of the rIGF-II and and were and of were of the the mitogenic and N-terminal to of the deletion mutants the to to rIGF-II and were for binding to insulin and and and were for its The and and were in binding to The and The of these in the were by these the of the the the of the N-terminal and of its binding affinity for IGF and insulin were the affinity of the N-terminal deletion mutants of rIGF-II for the and insulin in and and the were the affinity of the mutant of of The in the affinity of the deletion mutants for the in the of N-terminal by the in to in to by in of the binding of and to and the of for mutants of insulin and to the affinity of for IGF to of of the mutant for IGF and insulin of of to the of the for the and to the insulin and of deletion mutants deletion in the to affinity to rIGF-II the deletion of the the and in to the and insulin and of the of of The affinity of for of receptors, and and affinity for the were in binding and the role the in in its for and insulin the of by by to the of and and the of and of the binding the and the The and and to the N-terminal of in the binding of the growth to the and insulin and the affinity to the N-terminal to the of the of in the of affinity binding of the of the mutant to its and the The of these of of rIGF-II to of the for and in in in in in in in in and the in The and of rIGF-II and in affinity of for the and the of the N-terminal the affinity to these the for were of of The and The of the of to in of and were and in the activity of and The to role the the in the binding of and for the IGF and insulin to for and in binding to the insulin the in affinity of and for the and to the of the of of for its binding to the and growth growth insulin-like growth growth insulin-like growth and of and and the of the of and and the The and of and by and the and binding of the insulin-like growth insulin-like growth insulin-like growth insulin-like growth and affinity to receptors, and IGF-II, and to affinity to the and insulin of its affinity binding to the for binding to the and site-directed and in the and were for binding to the insulin and receptors, and in the were for its binding to these in and in the in and were for the binding of to The of the N-terminal of for of mitogenic activity in and and The in mitogenic to the of in the affinity of for the growth to To the of of deletion mutants of the N-terminal of rIGF-II prepared by and affinity for affinity for the the affinity of for IGF and of of and the affinity of mutants for IGF and insulin of of The and The of rIGF-II by the N-terminal region the prepared of deletion mutants and substitution mutant in the N-terminal to define the region of the to the binding of rIGF-II to and and were and rIGF-II were the of rIGF-II to the affinity of binding of to prepared by the insulin and were were of the and were to The The to the prepared The by affinity and to to insulin and affinity The of the by by were by and by the of rIGF-II for in Escherichia in the of The N-terminal substitution deletion mutants of rIGF-II were by site-directed the in The substitution mutant prepared the of the mutant by its affinity to the insulin the of the N-terminal deletion des-(1-5)-, des-(1-7)-, and N-terminal by and of mutants were expressed in of these were by The mutant were and the were by of the rIGF-II mutants for by and and N-terminal of the in the the by to were by the were for rIGF-II and of in and and in the were to of and were and of were to the of of rIGF-II to and for the binding of and to insulin and by and rIGF-II mutants were of of were in of of and of were by the of of and of were and in of the mutants for were by prepared of were in of of of and of The of the mutants for were were in of of of and of were by the were and to were the the of in the of of and the of in the of The for the binding of these to the were the of of of the of rIGF-II of rIGF-II mutants were by the of of were in were in of rIGF-II rIGF-II mutants in were to the in for the of the were for of and by The of by and were and rIGF-II were the of rIGF-II to the affinity of binding of to prepared by the insulin and were were of the and were to The The to the prepared The by affinity and to to insulin and affinity The of the by by were by and by the of rIGF-II for in Escherichia in the of The N-terminal substitution deletion mutants of rIGF-II were by site-directed the in The substitution mutant prepared the of the mutant by its affinity to the insulin the of the N-terminal deletion des-(1-5)-, des-(1-7)-, and N-terminal by and of mutants were expressed in of these were by The mutant were and the were by of the rIGF-II mutants for by and and N-terminal of the in the the by to were by the were for rIGF-II and of in and and in the were to of and were and of were to the of of rIGF-II to and for the binding of and to insulin and by and rIGF-II mutants were of of were in of of and of were by the of of and of were and in of the mutants for were by prepared of were in of of of and of The of the mutants for were were in of of of and of were by the were and to were the the of in the of of and the of in the of The for the binding of these to the were the of of of the of rIGF-II of rIGF-II mutants were by the of of were in were in of rIGF-II rIGF-II mutants in were to the in for the of the were for of and by The of by and were and rIGF-II were the of rIGF-II to the affinity of binding of to prepared by the insulin and were were of the and were to The The to the prepared The by affinity and to to insulin and affinity The of the by by were by and by the and were and rIGF-II were the of rIGF-II to the affinity of binding of to prepared by the insulin and were were of the and were to The The to the prepared The by affinity and to to insulin and affinity The of the by by were by and by the of rIGF-II for in Escherichia in the of The N-terminal substitution deletion mutants of rIGF-II were by site-directed the in The substitution mutant prepared the of the mutant by its affinity to the insulin the of the N-terminal deletion des-(1-5)-, des-(1-7)-, and N-terminal by The in the of The N-terminal substitution deletion mutants of rIGF-II were by site-directed the in The substitution mutant prepared the of the mutant by its affinity to the insulin the of the N-terminal deletion des-(1-5)-, des-(1-7)-, and N-terminal by and of mutants were expressed in of these were by The mutant were and the were by of the rIGF-II mutants for by and and N-terminal of the in the the by to were by the were for rIGF-II mutants were expressed in of these were by The mutant were and the were by of the rIGF-II mutants for by and and N-terminal of the in the the by to were by the were for rIGF-II and of in and and in the were to of and were and of were to the rIGF-II and of in and and in the were to of and were and of were to the of of rIGF-II to and for the binding of and to insulin and by and rIGF-II mutants were of of were in of of and of were by the of of and of were and in of the mutants for were by prepared of were in of of of and of The of the mutants for were were in of of of and of were by the were and to were the the of in the of of and the of in the of The for the binding of these to the were the of of for the binding of and to insulin and by and rIGF-II mutants were of of were in of of and of were by the of of and of were and in The of the mutants for were by prepared of were in of of of and of The of the mutants for were were in of of of and of were by the were and The to were the the of in the of of and the of in the of The for the binding of these to the were the of of of the of rIGF-II of rIGF-II mutants were by the of of were in were in of rIGF-II rIGF-II mutants in were to the in for the of the were for of and by The of by The of rIGF-II mutants were by the of of were in were in of rIGF-II rIGF-II mutants in were to the in for the of the were for of and by The of by of rIGF-II N-terminal deletion des-(1-5)-, des-(1-7)-, and and the substitution mutant were prepared and by and The of the and and and and in the deletion mutants were by and the mutant to The N-terminal for des-(1-5)-, des-(1-7)-, and were by to and The of the and of these mutants in of des-(1-7)-, and mutants were of the rIGF-II in The were of the mutants were of the of rIGF-II and rIGF-II and of of rIGF-II rIGF-II and were in of rIGF-II for to and for the binding of insulin and rIGF-II to insulin were and The for the binding of and rIGF-II to were and to in for these The of for rIGF-II binding to the in in the the of to of the in to the of affinity of binding of mutant to the in and of to of the binding of rIGF-II mutants to and of binding to insulin and by insulin rIGF-II and The the of to and the of in the of of and the of in its in The deletion of the and the N-terminal of the the of in the affinity of the rIGF-II mutants for the insulin and and to the insulin and and of the affinity of rIGF-II the to the the binding of the mutants to the the of des-(1-5)-, and for the were and of of to the of N-terminal of its binding to IGF the for the binding of rIGF-II to of the in the to the in the The of and for the were and of of rIGF-II The affinity of the for the for the for the affinity of the for of the of binding of the mutants to and deletion of the N-terminal the affinity of the mutants for the the binding of the rIGF-II to for the N-terminal to The binding of and of of these to for The of its to to the the binding affinity of the mutant by to of the the and of by and of the of rIGF-II the of in to the and for the binding of to were for the of the the of of of the N-terminal deletion mutants of rIGF-II the of the rIGF-II and and were and of were of the the mitogenic and N-terminal to of the deletion mutants the to to of rIGF-II N-terminal deletion des-(1-5)-, des-(1-7)-, and and the substitution mutant were prepared and by and The of the and and and and in the deletion mutants were by and the mutant to The N-terminal for des-(1-5)-, des-(1-7)-, and were by to and The of the and of these mutants in of des-(1-7)-, and mutants were of the rIGF-II in The were of the mutants were of the of rIGF-II for to and for the binding of insulin and rIGF-II to insulin were and The for the binding of and rIGF-II to were and to in for these The of for rIGF-II binding to the in in the the of to of the in to the of affinity of binding of mutant to the in and of to of the binding of rIGF-II mutants to and of binding to insulin and by insulin rIGF-II and The the of to and the of in the of of and the of in its in The deletion of the and the N-terminal of the the of in the affinity of the rIGF-II mutants for the insulin and and to the insulin and and of the affinity of rIGF-II the to the the binding of the mutants to the the of des-(1-5)-, and for the were and of of to the of N-terminal of its binding to IGF the for the binding of rIGF-II to of the in the to the in the The of and for the were and of of rIGF-II The affinity of the for the for the for the affinity of the for of the of binding of the mutants to and deletion of the N-terminal the affinity of the mutants for the the binding of the rIGF-II to for the N-terminal to The binding of and of of these to for The of its to to the the binding affinity of the mutant by to of the the and of by and of the of rIGF-II the of in to the and for the binding of to were for the of the the of of of the N-terminal deletion mutants of rIGF-II the of the rIGF-II and and were and of were of the the mitogenic and N-terminal to of the deletion mutants the to to of rIGF-II N-terminal deletion des-(1-5)-, des-(1-7)-, and and the substitution mutant were prepared and by and The of the and and and and in the deletion mutants were by and the mutant to The N-terminal for des-(1-5)-, des-(1-7)-, and were by to and The of the and of these mutants in of des-(1-7)-, and mutants were of the rIGF-II in The were of the mutants were of the The N-terminal deletion des-(1-5)-, des-(1-7)-, and and the substitution mutant were prepared and by and The of the and and and and in the deletion mutants were by and the mutant to The N-terminal for des-(1-5)-, des-(1-7)-, and were by to and The of the and of these mutants in of des-(1-7)-, and mutants were of the rIGF-II in The were of the mutants were of the of rIGF-II for to and for the binding of insulin and rIGF-II to insulin were and The for the binding of and rIGF-II to were and to in for these The of for rIGF-II binding to the in in the the of to of the in to the of affinity of binding of mutant to the in and of to in The deletion of the and the N-terminal of the the of in the affinity of the rIGF-II mutants for the insulin and and to the insulin and and of the affinity of rIGF-II the to the the binding of the mutants to the the of des-(1-5)-, and for the were and of of to the of N-terminal of its binding to IGF the for the binding of rIGF-II to of the in the to the in the The of and for the were and of of rIGF-II The affinity of the for the for the for the affinity of the for of the of binding of the mutants to and deletion of the N-terminal the affinity of the mutants for the the binding of the rIGF-II to for the N-terminal to The binding of and of of these to for The of its to to the the binding affinity of the mutant by to of the the and of by and The for the binding of insulin and rIGF-II to insulin were and The for the binding of and rIGF-II to were and to in for these The of for rIGF-II binding to the in in the the of to of the in to the of affinity of binding of mutant to the in and of to The deletion of the and the N-terminal of the the of in the affinity of the rIGF-II mutants for the insulin and and to the insulin and and of the affinity of rIGF-II the to the the binding of the mutants to the the of des-(1-5)-, and for the were and of of to the of N-terminal of its binding to IGF the for the binding of rIGF-II to of the in the to the in the The of and for the were and of of rIGF-II The affinity of the for the for the for the affinity of the for of the of binding of the mutants to and deletion of the N-terminal the affinity of the mutants for the The the binding of the rIGF-II to for the N-terminal to The binding of and of of these to for The of its to to the the binding affinity of the mutant by to of the the and of by and of the of rIGF-II the of in to the and for the binding of to were for the of the the of of of the N-terminal deletion mutants of rIGF-II the of the rIGF-II and and were and of were of the the mitogenic and N-terminal to of the deletion mutants the to to the of in to the and for the binding of to were for the of the the of of of the N-terminal deletion mutants of rIGF-II the of the rIGF-II and and were and of were of the the mitogenic and N-terminal to of the deletion mutants the to to rIGF-II and were for binding to insulin and and and were for its The and and were in binding to The and The of these in the were by these the of the the the of the N-terminal and of its binding affinity for IGF and insulin were the affinity of the N-terminal deletion mutants of rIGF-II for the and insulin in and and the were the affinity of the mutant of of The in the affinity of the deletion mutants for the in the of N-terminal by the in to in to by in of the binding of and to and the of for mutants of insulin and to the affinity of for IGF to of of the mutant for IGF and insulin of of to the of the for the and to the insulin and of deletion mutants deletion in the to affinity to rIGF-II the deletion of the the and in to the and insulin and of the of of The affinity of for of receptors, and and affinity for the were in binding and the role the in in its for and insulin the of by by to the of and and the of and of the binding the and the The and and to the N-terminal of in the binding of the growth to the and insulin and the affinity to the N-terminal to the of the of in the of affinity binding of the of the mutant to its and the The of these in affinity of for the and the of the N-terminal the affinity to these the for were of of The and The of the of to in of and were and in the activity of and The to role the the in the binding of and for the IGF and insulin to for and in binding to the insulin the in affinity of and for the and to the of the of of for its binding to the and rIGF-II and were for binding to insulin and and and were for its The and and were in binding to The and The of these in the were by these the of the the the of the N-terminal and of its binding affinity for IGF and insulin were the affinity of the N-terminal deletion mutants of rIGF-II for the and insulin in and and the were the affinity of the mutant of of The in the affinity of the deletion mutants for the in the of N-terminal by the in to in to by in of the binding of and to and the of for mutants of insulin and to the affinity of for IGF to of of the mutant for IGF and insulin of of to the of the for the and to the insulin and of deletion mutants deletion in the to affinity to rIGF-II the deletion of the the and to the and insulin and of the of of The affinity of for of receptors, and and affinity for the were in binding and the role the in in its for and insulin the of by by to the of and and the of and of the binding the and the The and and to the N-terminal of in the binding of the growth to the and insulin and the affinity to the N-terminal to the of the of in the of affinity binding of the of the mutant to its and the The of these The in affinity of for the and the of the N-terminal the affinity to these the for were of of The and The of the of to in of and were and in the activity of and The to role the the in the binding of and for the IGF and insulin to for and in binding to the insulin the in affinity of and for the and to the of the of of for its binding to the and to and for the of to for and and for