Structural plasticity of an invariant hydrophobic triad in the switch regions of Rab GTPases is a determinant of effector recognition.

Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J.

The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding. Critical role of this dual interaction for endosomal localization.

Early endosome antigen 1 (EEA1) is 170-kDa polypeptide required for endosome fusion. EEA1 binds to both phosphtidylinositol 3-phosphate (PtdIns3P) and to Rab5-GTP in vitro, but the functional role of this dual interaction at the endosomal membrane is unclear. Here we have determined the structural features in EEA1 required for binding to these ligands.

Interaction of insulin receptor substrate-1 with the sigma3A subunit of the adaptor protein complex-3 in cultured adipocytes.

Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors.

Association of acyl-CoA synthetase-1 with GLUT4-containing vesicles.

GLUT4, the glucose transporter present in insulin-sensitive tissues, resides in intracellular vesicular structures and translocates to the cell surface in response to insulin. In an attempt to identify proteins present in these structures, GLUT4-enriched vesicles prepared from rat adipocytes treated with or without insulin were prepared by sucrose velocity gradient centrifugation and immunoadsorbed with anti-GLUT4 antibody. We report here the sequence identification by high performance liquid chromatography-ion trap mass spectrometry of a p75 protein band, long chain acyl-CoA synthetase-1, specifically present in immunoadsorbed GLUT4-containing vesicles but not in vesicles adsorbed by nonimmune serum.

Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles.

Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R.

Insulin regulation of membrane-associated insulin receptor substrate 1.

Insulin stimulation of 3T3-L1 adipocytes results in rapid activation of the insulin receptor tyrosine kinase followed by autophosphorylation of the receptor and phosphorylation of insulin receptor substrate 1 (IRS-1), its major substrate. The insulin receptor resides mostly at the cell surface of 3T3-L1 adipocytes under basal conditions, while about two-thirds of IRS-1 fractionates with intracellular membranes and one-third fractionates with cytosol. To test whether insulin receptor internalization is required for optimal tyrosine phosphorylation of IRS-1, 3T3-L1 adipocytes and CHO-T cells were incubated at 4 degrees C which inhibits receptor endocytosis but not its tyrosine kinase activity.

The bradykinin analog RMP-7 increases intracellular free calcium levels in rat brain microvascular endothelial cells.

The vasoactive peptide bradykinin is believed to cause increased vascular permeability by the activation of B2 receptors on the vascular endothelium. A bradykinin analog, H-Arg-Pro-Hyp-Gly-Thi-Ser-Pro-4-Me-Tyr(psi CH2NH)-Arg-OH (RMP-7), was designed and it was proposed that it might increase cerebrovascular permeability by activating B2 receptors on brain microvasculature. In this report, the effects of RMP-7 and related peptides on bradykinin receptor-induced calcium signaling were examined in rat brain microvascular endothelial (RBME) cultures.

FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains.

Myogenin belongs to a family of myogenic helix-loop-helix (HLH) proteins that activate muscle transcription through binding to a conserved DNA sequence associated with numerous muscle-specific genes. Fibroblast growth factor (FGF) inhibits myogenesis by inactivating myogenic HLH proteins. We show that activated protein kinase C (PKC) can substitute for FGF and inhibit transcriptional activity of myogenic HLH proteins.

Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators.

Enhanced casein kinase II activity in COS-1 cells upon overexpression of either its catalytic or noncatalytic subunit.

Casein kinase II consists of catalytic (alpha) and regulatory (beta) subunits complexed into a heterotetrameric alpha 2 beta 2 structure. Full-length cDNAs encoding the alpha and beta subunits of human casein kinase II were subcloned into an expression vector containing the cytomegalovirus promotor, yielding the expression constructs pCMV-alpha and pCMV-beta. Northern analyses of total cellular RNA prepared from COS-1 fibroblasts 65 h after transfection with pCMV-alpha or pCMV-beta or with both expression constructs showed marked specific increases in corresponding alpha and beta subunit RNAs.

Rhodopsin expressed in Chinese hamster ovary cells regulates adenylyl cyclase activity.

The cDNA encoding bovine opsin was transfected into Chinese hamster ovary (CHO) cells to generate stable clones expressing the rod cell photoreceptor protein. Cells expressing opsin, when incubated in 11-cis retinal and exposed to light, inhibited forskolin-stimulated adenylyl cyclase activity. Rhodopsin-mediated inhibition of adenylyl cyclase was prevented by treatment of cells with pertussis toxin.

Cloning and characterization of a cDNA encoding the beta subunit of human casein kinase II.

Previous studies [Summercorn et al. (1987) Proc. Natl.

Molecular cloning of the human casein kinase II alpha subunit.

A human cDNA encoding the alpha subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.

Pepsin-generated type VI collagen is a degradation product of GP140.

A major extracellular matrix glycoprotein, GP140 , synthesized by WI-38 human lung fibroblasts has previously been shown to be collagen-like. A form of GP140 that is related to extracellular matrix GP140 both antigenically and in apparent molecular mass was isolated from human placenta. Types I-VI collagen were isolated from human tissues by limited pepsin digestion, selective salt precipitation, and chromatography.