Burgess J W, Wada I, Ling N, Khan M N, Bergeron J J, Posner B I
Polypeptide Hormone Laboratory, Royal Victoria Hospital, Montreal, Canada.
J Biol Chem. 1992 May 15;267(14):10077-86.
In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.
在先前的一项研究中，我们发现，胰岛素注射后，大鼠肝内体（EN）的胰岛素受体（IR）激酶会短暂激活，其水平超过质膜（PM）受体。用磷酸酶处理EN受体可消除IR激酶的激活，这表明β亚基的自磷酸化是激活过程的介质（Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931 - 12940）。在本研究中，通过两种不同方法估算了PM和EN中IRβ亚基的磷酸酪氨酸（PY）含量。一种方法是用32Pi进行直接体内标记，随后进行受体免疫沉淀。第二种方法是，在注射胰岛素后，用针对IRβ亚基跨膜结构域（包含960位残基（α960））的抗体和针对PY的抗体（αPY）进行免疫印迹，以测定PM和EN中PY/β亚基的含量。通过这两种方法均发现，PM中IR的PY含量显著高于EN中IR的PY含量。给予1.5微克胰岛素/100克体重（50%受体占有率）或15微克/100克体重（受体饱和）的剂量时，PM中IR的PY/β亚基水平达到EN中IR的2.0至2.5倍。令人惊讶的是，自磷酸化后，EN中的IR比PM中的IR掺入的PY/β亚基多3至5倍。PM中的外源性IR激酶活性（聚（Glu:Tyr））随胰岛素剂量变化很小。相比之下，EN受体的激酶活性呈剂量依赖性增加，同时PY/β亚基水平降低。对自磷酸化前后αPY免疫沉淀的受体和激酶活性比例进行比较表明，EN中而非PM中含有一小部分轻度磷酸化但高度活化的受体。由于Thr12 - Lys（IR激酶残基1142 - 1153）可有效抑制PM和EN受体的IR自磷酸化，因此β亚基调节酪氨酸的Tris磷酸化不太可能。这些结果可能可以通过IR激酶的去磷酸化依赖性激活来解释，就像酪氨酸激酶的src家族那样。
