Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF.

363

331

Caveolae and detergent-insoluble, glycosphingolipidenriched domains (DIGs) are cholesterol-enriched membrane domains that have been implicated in signal transduction because a variety of signaling proteins as well as phosphatidylinositol bisphosphate (PtdInsP 2 ) are compartmentalized in these domains. We report here that depletion of cellular cholesterol leads to the inhibition of epidermal growth factor-and bradykininstimulated PtdIns turnover in A431 cells. This is associated with the loss of compartmentalization of epidermal growth factor receptors, G q , and PtdInsP 2 in the low density membrane domains. Replacement of cellular cholesterol leads to the reorganization of signaling molecules in the low density domains and the reestablishment of hormone-stimulated PtdIns hydrolysis. Oxysterol derivatives show a variable ability to functionally replace the cholesterol in this system. These data are consistent with the hypothesis that localization of signaling proteins and lipids to cholesterol-enriched domains is required for the proper function of hormonestimulated PtdIns turnover.

Section: Methodsmentioning

confidence: 99%

Cholesterol Depletion Delocalizes Phosphatidylinositol Bisphosphate and Inhibits Hormone-stimulated Phosphatidylinositol Turnover

Pike

Miller

1998

363

331

“…Anti-actin antibodies were from Chemicon (El Segundo, CA). The polyclonal anti-EGF receptor antibody, DB1, was described previously (15 (16). All other chemicals were from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Localization and Turnover of Phosphatidylinositol 4,5-Bisphosphate in Caveolin-enriched Membrane Domains

Pike

Casey

1996

341

239

Caveolae are small, plasma membrane invaginations that have been implicated in cell signaling. In A431 cells, approximately half of the total cellular phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ) was found to be localized in low density, Triton-insoluble membrane domains enriched in caveolin. Treatment of cells with either epidermal growth factor or bradykinin for 5 min at 37°C resulted in approximately a 50% decrease in this caveolar PtdIns 4,5-P 2 with no change in the levels of plasma membrane PtdIns 4,5-P 2 . These data suggest that the PtdIns 4,5-P 2 present in cells is largely compartmentalized and that the caveolar PtdIns 4,5-P 2 is subject to hydrolysis by hormone-stimulated phospholipase C. As growth factor receptors, seven transmembrane domain receptors, heterotrimeric G proteins, and the inositol trisphosphate receptor have all been shown to be enriched in caveolae, these findings suggest that both the generation and response to inositol trisphosphate is highly compartmentalized within the cell.Caveolae are small, plasma membrane invaginations that are involved in apical protein sorting (1, 2) and the uptake of folates by potocytosis (3). In most cell types, they account for 1% or less of the total plasma membrane (4). Caveolin, a 21-kDa substrate for pp60 src (5), is localized almost exclusively to caveolae and probably represents a structural component of this plasma membrane domain (6 -8). Caveolae are also enriched in glycosphingolipids and cholesterol, making these domains resistant to extraction in Triton X-100 (1).Caveolae have been shown to contain a variety of molecules involved in cell signaling including low molecular weight and heterotrimeric G proteins (9, 10), Src family kinases (10), mitogen-activated protein kinase (10), the epidermal growth factor receptor (11,12), and the platelet-derived growth factor receptor (13). These findings implicate caveolae in signal transduction and suggest that many of the molecular components for cell signaling are localized in this relatively small area of the plasma membrane.We have recently shown that in Madin Darby canine kidney cells, a large proportion of the total cellular PtdIns 4,5-P 2 1 resides in detergent-insoluble lipid domains enriched in caveolin (14). Given this observation, the question arises as to whether the PtdIns 4,5-P 2 present in caveolae is subject to turnover in response to growth factors and hormones. We now report that, in A431 cells, at least 50% of the PtdIns 4,5-P 2 is present in caveolae, and this pool of phospholipid is reduced in cells treated with EGF or bradykinin. Polyphosphoinositides present in the plasma membrane fraction are not altered by hormone treatment. These findings suggest that PtdIns 4,5-P 2 is highly compartmentalized within cells and that caveolae are the primary site of agonist-stimulated PtdIns 4,5-P 2 turnover. EXPERIMENTAL PROCEDURESMaterials-Anti-caveolin antibodies were from Transduction Laboratories (Lexington, KY). Anti-actin antibodies were from Chemicon (El Segundo, CA). The polyclo...

“…Antibodies to MAP kinase, FAK, Ras-GAP and PI 3-kinase were obtained from UBI. Polyclonal antibodies (DB-1) to the EGF receptor and 125 I-EGF were prepared as described previously (17). Arg-Arg-Src peptide (RRLIEDAEYAARG) was synthesized on an Applied Biosystems peptide synthesizer and purified by ion exchange and reversephase high pressure liquid chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…At the end of the incubation, cells were washed three times in Hanks' balanced buffer solution. Monolayers were dissolved by the addition of 1 ml of NaOH and cell associated 125 I-EGF determined by ␥ counting (17). Nonspecific binding was determined in duplicate wells containing 500 nM unlabeled EGF.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

See 1 more Smart Citation

The Multiple Endocrine Neoplasia Type 2B Point Mutation Alters Long-term Regulation and Enhances the Transforming Capacity of the Epidermal Growth Factor Receptor

Pandit

Donis-Keller

Iwamoto

et al. 1996

The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wildtype receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P ؉ 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.RET was first identified as a transforming gene by transfection experiments using DNA isolated from a human T-cell lymphoma (1). Subsequently, physical and genetic mapping data have implicated the RET gene in several dominantly inherited human neoplasias, including multiple endocrine neoplasia type 2A (MEN 2A), 1 multiple endocrine neoplasia type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) (2-6). MEN 2A and FMTC are both characterized by the development of medullary thyroid carcinoma between the ages of 20 and 50. Approximately one-third to one-half of MEN 2A patients also develop pheochromocytomas and parathyroid hyperplasia. MEN 2B is a more severe form of the disease with onset of symptoms during the first decade of life. Patients exhibit ganglioneuromas of the intestinal tract, mucosal neuromas, and opthalmalogic and skeletal abnormalities, in addition to a particularly aggressive form of medullary thyroid carcinoma.The RET proto-oncogene encodes a protein that is a member of the family of transmembrane tyrosine kinase growth factor receptors. The RET protein is comprised of a 635-amino acid extracellular domain, a single transmembrane spanning region, and an intracellular tyrosine kinase domain that contains a short, 26-amino acid kinase insert domain. Residues 515 to 634 of the extracellular domain constitute a region of high cysteine content similar to those found in the EGF receptor and the insulin receptor (7, 8). Immediately amino-terminal to this high cysteine region is a domain of ϳ110 amino acids that is 20 -30% homologous to cadherins (9) The presence of this cadherin-like domain is unique among ...