Abstract
Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma-32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10-fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P-labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the Department of Biochemistry, University of Massachusetts Medical Center, Worcester, Massachusetts 01605 and $Amgen Devdopment, Inc., Boulder, Colorado 80301
Insulin-likegrowthfactor I (IGF-I)receptors are the trypsinizedIGF-I receptor @ subunits are chromatpartially purified from human placentaby sequential ographically identical to those from the tryptic hyaffinity chromatography with wheat germ agglutinin-drolysates of 32P-labeled insulin receptor @ subunit
We demonstrate that like the insulin receptor kinase [26,27,28] the IGF-I receptor-associated kinase activity is dramatically activated by prior phosphorylation of its fi subunit on tyrosine residues' or by treatment with reductant
Summary
Apart from a major protein of M , 100,000, the amounts of these contaminants as estimated by the intensity of silver staining are relatively minor when compared to that of the IGF-I receptor a subunit These contaminants appear to adsorb to thIeGF-I*-agarose matrix nonspecifically, too IO 100 IO and their adsorptionis not dependent on the presence of the nM IGF-I nM I N S. The degrees of contamination by these protein species as evidenced by the silver staining patterns are notdiminished when the IGF-Ireceptor is preventedfrom binding to the IGF-I*-agarose in the presence of excess free. When such a preparation of immobilized IGF-I receptoris first incubated in the presence of increasing concentrations of ATP for 15min at 22 "Cfollowed by extensivewashing to remove theunreactedATP,the histone kinase activityof the IGF-Ireceptor preparation from theATP-treated group is progressively increased (Fig. 2)
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