Insulin-like growth factor 1 receptor-mediated endocytosis in Xenopus laevis oocytes. A role for receptor tyrosine kinase activity.
Equilibrium analysis of [125I]IGF-1 binding to Xenopus oocytes yielded curvilinear Scatchards supporting the presence of multiple binding sites. High-affinity binding displayed an apparent KD of 9.0 +/- 3.0 nM IGF-1 with maximum total binding of 1.6 +/- 0.3 fmole/oocyte after 90 min at 22 degrees C. Binding of 1 nM [125I]IGF-1 to oocytes was competitively displaced by unlabeled IGF-1 or insulin with IC50 values of 6 and 560 nM, respectively. Chemical cross-linking of [125I]-IGF-1 to oocyte membranes specifically labeled a single 138-kDa protein. Using the technique of acid stripping to analyze whole-cell binding, both total and surface binding of [125I]IGF-1 reached a plateau after 90 min, and internalized counts surpassed surface-associated counts 20 min after hormone addition. At the 90-min plateau, 70-75% of cell-associated counts were internalized. The tyrosine kinase inhibitor, tyrphostin 47, prevented IGF-1-stimulated oocyte maturation with an apparent IC50 of 10.5 +/- 2.5 microM and also inhibited IGF-1 receptor-mediated endocytosis in micromolar concentrations. Treatment of oocyte membrane samples with 10 nM IGF-1 stimulated alkali-stable phosphorylation of a protein doublet (M(r) 89 and 104 kDa). Tyrphostin 47 also inhibited IGF-1-stimulated phosphorylation of these putative receptor beta-subunits more effectively than tyrphostin 23 or genistein. These results suggest a necessary role for receptor-mediated endocytosis in IGF-1-induced oocyte maturation and a requirement for receptor kinase activity in oocyte receptor internalization.
Taghon
M S
MS
Department of Biological Sciences, University of Denver, Colorado 80208.
Sadler
S E
SE
D000818:Animals; D004705:Endocytosis; D005260:Female; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000502:physiology*; D017526:Receptor, IGF Type 1 / Q000502:physiology*; D014982:Xenopus laevis
D007334:Insulin-Like Growth Factor I; D020794:Receptor Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1