Autophosphorylation within insulin receptor beta-subunits can occur as an intramolecular process.
The insulin receptor is a complex membrane-spanning glycoprotein composed of two alpha-subunits and two beta-subunits connected to form an alpha 2 beta 2 holoreceptor. Insulin binding to the extracellular alpha-subunits activates intracellular beta-subunit autophosphorylation and substrate kinase activity. The current study was designed to differentiate mechanisms of transmembrane signaling by the insulin receptor, specifically whether individual beta-subunits undergo cis- or trans-phosphorylation. We compared relative kinase activities of trypsin-truncated receptors, alpha beta-half receptors, and alpha 2 beta 2 holoreceptors under conditions that allowed us to differentiate intermolecular and intramolecular events. Compared to the insulin-stimulated holoreceptors, the trypsin-truncated receptor undergoes autophosphorylation at similar tyrosine residues and catalyzes substrate phosphorylation in the absence of insulin at a comparable rate. The truncated receptor sediments on a sucrose gradient at a position consistent with a structure comprising a single beta-subunit attached to a fragment of the alpha-subunit and undergoes autophosphorylation in this form in the absence of insulin. Autophosphorylation of the truncated insulin receptor is independent of receptor concentration, and immobilization of the truncated receptor on a matrix composed of an anti-receptor antibody bound to protein A-Sepharose diminishes neither autophosphorylation nor receptor-catalyzed substrate phosphorylation. Therefore, true intramolecular (cis) phosphorylations, which occur within individual beta-subunits derived from trypsin-truncated receptors, lead to kinase activation. However, insulin-stimulated autophosphorylation of insulin receptor alpha beta heterodimers is concentration-dependent, and both autophosphorylation and kinase activity are markedly reduced following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)
Shoelson
S E
SE
Research Division, Joslin Diabetes Center, Boston, MA 02215.
Boni-Schnetzler
M
M
Pilch
P F
PF
Kahn
C R
CR
D000818:Animals; D002460:Cell Line; D004800:Enzymes, Immobilized / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008114:Liver Neoplasms, Experimental; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin