Differential sensitivity of two functions of the insulin receptor to the associated proteolysis: kinase action and hormone binding.

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Abstract
Since we observed that after purification the receptor kinase activity is rapdily lost under conditions where insulin binding function seems to be preserved, we have studied the cause(s) of receptor kinase inactivation. Highly purified placental insulin receptor preparations were analyzed by NaDodSO4/PAGE followed by siliver staining or immunostaining using domain-specific antibodies raised against synthetic peptides corresponding to the amino acid sequences of the .beta. subunit. These studies revealed (i) the intact 90-kDa subunit is degraded first to an 88-kDa form and then to a 50-kDa .beta.1-subunit form by proteolysis even after purification when stored at 4.degree. C. (ii) The 88-kDa .beta. subunit, which lacks the carboxyl-terminal .apprxeq. 2-kDa portion exhibits almost no autophosphorylation activity, nor does insulin stimulate autophosphorylation. (iii) The loss of kinase activity as measured by phosphorylation of the src-related peptide is correlated with the loss of the intact 90-kDa .beta. subunit. (iv) Degradation of the .beta. subunit to the 50-kDa form seems to be facilitated by the removal of the .apprxeq. 2-kDa peptide. Present studies thus suggest that only the intact form of the .beta. subunit had full kinase activity in an insulin-dependent manner and that other forms, such as the 88-kDa .beta. subunit show little kinase activity. The inactivation appears to arise from a conformational change of the 90-kDa form, which makes it susceptible to proteolysis at the carboxyl-terminal end. These results imply that the carboxyl-terminal of the .beta. subunit is important for the manifestation of the tyrosine kinase activity of the insulin receptor.