Role of cholesterol in developing T-tubules: analogous mechanisms for T-tubule and caveolae biogenesis.

Recent work has suggested that caveolae biogenesis and transverse-tubule (T-tubule) formation in muscle cells share similar underlying features. We compared the properties of caveolin-1 (cav-1)-positive caveolae, in epithelial cells, with caveolin-3 (cav-3)-positive precursor T-tubules, in differentiating C2C12 muscle cells, using the cholesterol-binding drug, Amphotericin B (AmphB). Treatment of MDCK epithelial cells with acute high doses or chronic low doses of AmphB caused a loss of surface caveolae and the rapid redistribution of cav-1, and exogenously expressed cav-3, from the cell surface into modified endosomes. This effect was reversible and specific, as the GPI-anchored protein, alkaline phosphatase, was largely unaffected by the treatment unless it had been previously partitioned into caveolar domains. In differentiating C2C12 mouse myotubes, AmphB also caused a complete redistribution of cav-3 from precursor T-tubule elements into enlarged endosomes, morphologically very similar to those seen in MDCK cells. This was accompanied by redistribution of a T-tubule marker and a dramatic reduction in the extent of surface-connected tubular elements. We propose that cholesterol-enriched glycolipid 'raft' domains are involved in the formation and maintenance of diverse membrane systems including caveolae and the T-tubule system of muscle.

Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton.

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.

Constitutive expression of the orphan receptor, Rev-erbA alpha, inhibits muscle differentiation and abrogates the expression of the myoD gene family.

Rev-erbA alpha is an orphan steroid receptor that is expressed in skeletal muscle. Rev-erbA alpha binds to single/tandem copies of an AGGTCA motif, is transcribed on the noncoding strand of the c-erbA- alpha gene locus, and is postulated to modulate the thyroid hormone (T3) response. T3 induces terminal muscle differentiation and regulates fiber type composition via direct activation of the muscle-specific myoD gene family (e.g. myoD, myogenin). The myoD gene family can direct the fate of mesodermal cell lineages and activate muscle differentiation. Hence we investigated the expression and physiological role of Rev-erbA alpha during myogenesis. We observed abundant levels of Rev-erbA alpha mRNA in dividing C2C12 myoblasts, which were suppressed when the cells differentiated into postmitotic multinucleated myotubes. This decrease in Rev-erbA alpha mRNA correlated with the appearance of muscle-specific mRNAs (e.g. myogenin and alpha-actin). Constitutive overexpression of full length Rev-erbA alpha cDNA in the myogenic cells completely abolished differentiation, suppressed myoD mRNA levels, and abrogated the induction of myogenin mRNA. We then demonstrated that 1) GAL4-REV-erbA alpha chimeras that contain the 'AB' region and lack the 'E' region activated transcription of GAL4 response elements in the presence of 8-Br-cAMP and 2) the ligand-binding domain (LBD) contains an active transcriptional silencer. Overexpression of Rev-erbA alpha (delta AB) in myogenic cells had no impact on the ability of these cells to morphologically or biochemically differentiate. Furthermore, this orphan receptor 1) down-regulated thyroid hormone receptor (TR)/T3 mediated transcriptional activity from the myogenin promoter and thyroid hormone response element (TRE) an 2) disrupted TR homodimer and TR/retinoid X receptor (RXR) heterodimer formation on a number of TREs found in the myoD gene family. In conclusion, Rev-erbA alpha functions as a negative regulator of myogenesis by targeting the expression of the myoD gene family. The mechanism of action may involve inhibition of functional TR/RXR heterodimer formation on critical TREs and dominant trans-repression of gene expression.

Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.

Identification, purification and characterization of a novel phosphatidylinositol-specific phospholipase C, a third member of the beta subfamily.

The partial sequence of a novel PtdIns-specific phospholipase C of the beta subfamily (PtdIns-PLC beta 3) is described. Based upon the predicted protein sequence, monospecific antibodies have been raised and used to identify a suitable source for purification of the protein. Fractionation of HeLa S3 cells revealed that immunoreactive PtdIns-PLC beta 3 is membrane associated; purification (approximately 1000-fold) from this fraction yielded a single immunoreactive protein of 158 kDa, with a specific activity of 136 mumol.min-1.mg-1, with PtdIns 4,5-bisphosphate as substrate. Substrate specificity and Ca2+ dependence of this purified PtdIns-PLC are characteristic of the PtdIns-PLC beta subfamily.