Internalization of the constitutively active arginine 1152-->glutamine insulin receptor occurs independently of insulin at an accelerated rate.
Signals controlling the insulin receptor endocytotic pathway have been investigated using the R1152Q insulin receptor mutant (M). This mutant receptor exhibits high levels of insulin-independent kinase activity, impaired autophosphorylation, and lack of an insulin stimulatory effect on both auto- and substrate phosphorylation. NIH-3T3 fibroblasts expressing M receptors displayed a 2.5-fold higher 125I-insulin internalization rate than wild type (WT) but lacked insulin-induced receptor internalization and down-regulation. Cell surface recycling of internalized receptors also occurred at a higher rate in M cells and was unaffected by insulin. Cell preincubation with 35 mM Tris, which inhibits the insulin receptor degradative route, elicited no effect on M receptor recycling but inhibited that of WT by 40%. In contrast, the energy depleter 2,4-dinitrophenol, which inhibits normal insulin receptor retroendocytosis, impaired M receptor recycling 4-fold more effectively than that of WT. The release of internalized intact 125I-insulin was 6-fold greater in M than in WT fibroblasts and was almost completely inhibited by dinitrophenol, whereas insulin degradation by M cells was 4-fold decreased as compared with WT. Thus, internalization and recycling of the constitutively active Gln1152 receptor kinase occur in the absence of autophosphorylation. However, tyrosine phosphorylation appears to be required for proper sorting of endocytosed insulin receptors.
Formisano
P
P
Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, University of Naples Medical School, Italy.
DeNovellis
G
G
Miele
C
C
Tripodi
F
F
Caruso
M
M
Palumbo
G
G
Beguinot
L
L
Beguinot
F
F
D019297:2,4-Dinitrophenol; D016475:3T3 Cells; D000818:Animals; D001120:Arginine / Q000235:genetics; D004140:Dinitrophenols / Q000494:pharmacology; D015536:Down-Regulation; D004705:Endocytosis / Q000187:drug effects; D005973:Glutamine / Q000235:genetics; D007328:Insulin / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D014325:Tromethamine / Q000494:pharmacology
D004140:Dinitrophenols; D007328:Insulin; D014325:Tromethamine; D005973:Glutamine; D001120:Arginine; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D019297:2,4-Dinitrophenol