Receptor-mediated endocytosis of Au₁₆-hCG by porcine luteal cells

Author

Lisa Leigh Satterwhite

Date of Award

3-1984

Degree Type

Thesis

Degree Name

Master of Science

Major

Zoology

Major Professor

John H. Abel

Committee Members

R. Bagby, T. Chen

Abstract

Although the plasma membrane of porcine luteal ceils from a variety of species has been shown to be actively involved in the binding and internalization of human chorionic gonadotropin (hCG), the intracellular movement and fate of the hormone-receptor complex is not well understood. Previously, internalization of labeled hormone and a variety of membrane constituents was thought to occur in luteal cells in vivo via coated pits that transferred their contents to secondary lysosomes. Rarely was tracer observed between the cell surface and lysosomes except in a series of tenuous smooth surfaced canaliculi positioned directly beneath the plasma membrane. In order to more closely examine endocytosis of hormone by luteal cells in vitro a series of labeling experiments were initiated where cells were allowed to internalize a well characterized complex of Au₁₆-hCG. Porcine luteal cells, cultured following collagenase dispersal, were shown to bind ¹²⁵l-hCG. Binding was both specific, as indicated by the effective cold competition by native hormone, and saturable after 60 min at 37°C. Luteal cells that were incubated with Au₁₆-hCG for 4 min. at 4°C, and processed for electron microscopy, were observed to bind Au₁₆hCG diffusely to the microvillar extensions along the cell surface. Following warming to 37°C, Au₁₆-hCG was observed to form small clusters along the cell surface, and to be internalized in coated vesicles. Following endocytosis, gold beads were observed in smooth surfaced vesicles termed initial endosomes that were located directly beneath the plasma membrane. Multivesicular bodies (mvb) distributed near the perimeters of the cells also were observed to accumulate gold particles. After 90 min., gold beads were present in secondary endosomes located deep within the centrospheric regions of the cell, directly adjacent to Golgi elements and GERL.

The binding and internalization of Au₁₆-hCG appeared to be receptormediated because a) Au₁₆-BSA was not observed to bind luteal cells at 4°C, and associated with surface membrane minimally after 90 min., b) internalization of surface associated Au₁₆-hCG did not occur at 4°C, and c) binding of Au₁₆-hCG was blocked by incubation of cells with Au₁₆-hCG in the presence of excess native hCG.

Prior to any labeling experiments the gold complex was tested for the ability to bind the hCG receptor. Au₁₆-hCG was shown to effectively compete with ¹²⁵|-hCG for binding sites on membranes from corpora lutea, and to bind with an efficiency approximately one magnitude less than that observed for native hCG. In addition, Au₁₆-hCG was shown to elicit progesterone release from luteal cells that was comparable to release stimulated by native hormone.

In an attempt to examine microfilament redistribution concomitant with the endocytosis of hCG, myosin subfragment-l (S-l) was prepared by chymotryptic cleavage, purified by saturated ammonium sulfate precipitation, and shown to bind specifically to purified F-octin. Microfiloments from resting luteal cells were shown to decorate with S-l and to be distributed throughout the cytoplasm. Luteal cells incubated with Au₁₆-hCG for 60 min. at 37°C were subsequently labeled with S-l. Microfilaments that were labeled with SI were observed to associate directly with areas of the plasma membrane that were occupied by Au₁₆-hCG. Attempts to visualize the organization of microfiloments associated with intracellular organelles labeled with Au₁₆-hCG by decoration with S-l were unsuccessful. Microtubules were observed to be numerous along the endocytic pathway, intermediate filaments appeared to associate directly with coated vesicles containing Au₁₆-hCG and with lysosome like structures filled with gold beads.

In conclusion, Au₁₆-hCG was rapidly internalized by fully differentiated luteal cells along a pathway consistent with that observed during the receptor-mediated endocytosis of a variety of hormones and nutritive substances. We suggest that the initial endosomes observed to accumulate Au₁₆-hCG during endocytosis in vitro are the morphological equivalent to the tenuous processes that are observed to accumulate hormone in vivo. Cytoskeletol elements appeared to be directly associated with surface membranes and with membranes of subcellular organelles involved in the receptor-mediated endocytosis of Au₁₆-hCG. On the basis of these findings we plan to a) determine if the appearance of Au₁₆-hCG in initial endosomes prior to delivery to secondary endosomes is related to hormone and receptor uncoupling, and b) determine whether the repeated structural associations between cytoskeletol elements and organelles involved in the RME of Au₁₆-hCG are related functionally to the endocytic process.

Recommended Citation

Satterwhite, Lisa Leigh, "Receptor-mediated endocytosis of Au₁₆-hCG by porcine luteal cells. " Master's Thesis, University of Tennessee, 1984.
https://trace.tennessee.edu/utk_gradthes/14709