Abstract
We have investigated the vitellogenin (VTG) receptor system in Xenopus oocytes since these cells are specialized for endocytosis. Oocytes have between 0.2 and 3 X 10(11) receptors per 1-mm cell. There is only a single class of receptors of low affinity (1.3 X 10(-6) M at 22 degrees C and 2-4 X 10(-6) M at 0 degree C), but high specificity (less than 5% nonspecific binding at 2 X 10(-6) M). The specific internalization rate of the VTG receptor (around 2 X 10(-3) s-1) is first order, highly variable, and at the upper end of the range of values reported for mammalian cells. The receptor association rate constant (9.6 X 10(2) M-1 s-1) is extremely low although the dissociation rate constant was immeasurable. Calcium is required for VTG binding, and low pH does not dissociate the VTG-receptor complex. Monensin treatment at 100 microM caused the loss of surface receptors with a t1/2 of 3 h and the accumulation of internalized ligand in a "pre-lysosomal" endocytic compartment. Conversely, the recovery of surface VTG receptors that were removed with trypsin occurred with a t1/2 of about 2 h. These observations indicate that oocytes have very large intracellular pools of receptors and that although surface receptors are internalized on the time scale of minutes, the intracellular pool is recycled on the time scale of hours.
Highlights
Endocytosis in OocytesVitellogenin Labeling-VTG was phosphorylated in serum obtained from estrogen-stimulated animals (4 mg of estradiol/animal) using either Xenopus or chicken liver protein kinase [15]
Monensin inhibited VTG internalizationthrough its inhibitory effect on surface binding rather than affecting the specific internalization rate of the receptors, since this was not significantly different at 6 hpost-treatment (2.5 x s” versus 2.8 x s-’)
These results are consistent with the hypothesis that monensin prevents the recycling of VTG receptors
Summary
Vitellogenin Labeling-VTG was phosphorylated in serum obtained from estrogen-stimulated animals (4 mg of estradiol/animal) using either Xenopus or chicken liver protein kinase [15]. Binding and KineticAnalyses-The number of surface VTG receptors on oocytes was determined by Scatchard analyses [17] at 0 "C using VTG concentrations ranging from 6.5 X to 6.5 X M (0.03-3.0 mg/ml). Groups of 10 cells were incubated with the appropriate concentration of labeled VTG for 5 h at 0 "C. The ligands used were: VTG (0)p,hosvitin (01,heparin (W), casein (O), fetuin (A), and ovalbumin (A).The cells were rinsed, and the amount of associated radioactivity was determined as described under "Materials and Methods." The data were converted to a percentage of the radioactivity bound to the oocytes in the absence of any competing ligand. The cells were placed in prewarmed (22 "C) solution 0-R2 containing 10%calf serum and allowed to recover their surface receptors for varying amounts of time. Covered receptors was determined by incubating the oocytes for 5 h To determine the specificity of VTG binding, we incubated at 0 "C with 2.2 X M [32P]VTG
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