Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles.

Abstract

When '25I-labeled insulin ('25I-insulin) is incubated with 3T3-L1 adipocytes and cells processed for electron microscopic autoradiography, the ligand initially localizes preferentially to microvilli and coated pits. As a function of time and temperature, this initial preferential.localization to microvilli is lost, and the ligand is internalized by the cell. Serial sections of apparent coated vesicles near the cell surface indicate that about half of these structures are true vesicles and, therefore, intermediates in this receptor-mediated endocytotic process. With time, '251-insulin localizes to larger intracellular membranebounded structures. When cells are incubated with another ligand, cationic ferritin, that is taken up by adsorptive endocytosis, essentially the same structures are involved as for the endocytosis of '2I-insulin. The data suggest that specificity for receptor-mediated endocytosis is conferred by the. specific ligand receptor and possibly by ligand-induced receptor mobility in the plane of the plasma membrane. Other structures such as coated pits, coated vesicles, larger vesicles, and secondary lysosomes are common for different ligands.Direct morphologic studies have demonstrated that polypeptide hormones and their specific cell surface receptors are internalized by a variety of cell types (1-4). This receptor-mediated process provides a mechanism for removal of the ligand from the cell surface for inactivation and a mechanism by which cell surface receptors may be regulated.In the present investigation, we have tracked the morpho- with '25I-insulin (5 ng/ml) or cationic ferritin (0.75 mg/ml) for the times and temperatures specified. After the incubations, cells were washed in buffer at 4°C and fixed on the plate in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hr. The plates were then washed; and cells were postfixed in 0.1 M osmium tetroxide (pH 7.4), dehydrated in ethanol, and embedded in Epon. Thin sections were cut parallel to the plane of the culture and placed on copper grids.Grids containing '"I-insulin were then processed for autoradiography (6, 7).Sampling and Analysis of Data. The method ofsampling was similar to that as described (7,8). All The relationship ofautoradiographic grains to specific plasma membrane structures was assessed by the "hypothetical grain" method of Blackett and Parry (9, 10) and will be described in detail elsewhere (11) as applied to this type of study. In brief, the method analyzes the "real grains" (i.e., developed autoradiographic grains) in terms of their relationship to underlying structures versus the distribution of random or "hypothetical grains." If the real-grain distribution can be fitted to the hypothetical-grain distribution by a specially designed computer program, then the distribution is random. Failure ofreal grains to be fitted to the hypothetical-grain distribution represents preferential labeling ofa structure and is expressed by the term "grains per grid point. " The data also can be expressed in terms ofrelat...

The neck of caveolae is a distinct plasma membrane subdomain that concentrates insulin receptors in 3T3-L1 adipocytes

“…Other studies have also shown that the EGF receptor associates with caveolar-like domains for signaling, then exits these structures to be captured by clathrin-coated pits and internalized (23). Our data regarding the IRs support this concept because we found that activated IRs are recruited to the caveola neck microdomains upon insulin stimulation but are likely internalized through clathrin-coated pits as we previously demonstrated (24). Because many other lipid raft-dependent signaling pathways rely on raft aggregation, IR aggregation to the caveola neck microdomains might thus represent an important mechanism to mediate transduction of insulin signaling in 3T3-L1 adipocytes.…”

Insulin-induced surface redistribution regulates internalization of the insulin receptor and requires its autophosphorylation.

“…Fixed cells were dehydrated, processed for electron microscope (EM) autoradiography, and quantitated as described (1)(2)(3)12). Three experiments were performed.…”

“…We have shown that the initial binding of insulin to its receptor preferentially occurs on thin digitations of the cell surface, the microvilli (1-4). The insulin-receptor complex then redistributes in the plane of the membrane and concentrates on nonvillous segments from which it is internalized by pinching off the coated pits that form coated vesicles (1)(2)(3)(4). Through regulating the number of surface insulin receptors and by facilitating the intracellular degradation of insulin, stimulation of the internalization of the insulin receptor plays a key role in the biological action of the hormone.…”

Protein Kinase C-ζ and Protein Kinase B Regulate Distinct Steps of Insulin Endocytosis and Intracellular Sorting

“…In addition, hormone-bound IR is rapidly removed from the cell surface and internalized within clathrin-coated vesicles (3). Internalized insulin is then either degraded or released intact in the extracellular medium (4,5).…”