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PubMed1990

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.

Adenocarcinoma↗
PubMed1998

Intracellular nucleotide-mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil.

1. We have examined the properties of intracellular nucleotide-mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell-attached form, both types of K+ channel were activated by pinacidil. 2. In inside-out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi-mediated channel inhibition in a concentration-dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di- or tri-, but not mono-, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil. 3. In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell-shaped concentration-dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil. 4. From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide-independent fast channel gate and a nucleotide-dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate. 5. We conclude that Kir6.0 subunits play a crucial role in the nucleotide-mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.

Adenosine Diphosphate↗
PubMed1995

Interaction of the transforming growth factor-beta type I receptor with farnesyl-protein transferase-alpha.

Transforming growth factor-beta 1 (TGF-beta 1) is the prototype of a large family of molecules that regulate a variety of biological processes. The type I (T beta R-I) and type II (T beta R-II) receptors for TGF-beta 1 are transmembrane serine/threonine kinases, forming a heteromeric signaling complex. Recent studies have shown that T beta R-II is a constitutively active kinase and phosphorylates T beta R-I upon ligand binding, suggesting that T beta R-I is the effector subunit of the receptor complex, which transduces signals to intracellular targets. This model has been further confirmed by the identification of constitutively active T beta R-I that mediates TGF-beta 1-specific cellular responses in the absence of ligand and T beta R-II. To investigate signaling by TGF-beta 1, we have sought to isolate proteins that interact with the cytoplasmic region of T beta R-I. One of the proteins identified was the alpha subunit of farnesyl-protein transferase (FT alpha) that modifies a series of peptides including Ras. T beta R-I specifically interacts with FT alpha in the yeast two-hybrid system. Glutathione S-transferase-T beta R-I fusion proteins bind FT alpha translated in vitro. T beta R-I also phosphorylates FT alpha. We further show that the constitutively active T beta R-I interacted with FT alpha very strongly whereas an inactive form of T beta R-I did not. These results suggest that FT alpha may be one of the substrates of the activated T beta R-I kinase.

Activin Receptors, Type I↗
PubMed2003

Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD.

The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli. FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD(N)) comprising the N-terminal 139 residues of FimD. Purified FimD(N) is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD(N) retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC-FimH complex. Overexpression of FimD(N) in the periplasm of wild-type E.coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD(N) and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates.

Adhesins, Escherichia coli↗
PubMed1996

Adhesion molecules in human crescentic glomerulonephritis.

The expression of the intercellular adhesion molecule-1 (ICAM-1) and its ligand lymphocyte function associated antigen-1 (LFA-1 or alpha L), the vascular cell adhesion molecule-1 (VCAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1), and the cellular receptors for extracellular matrix, alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, alpha V, beta 1, and beta 3 integrin subunits, was studied in 28 patients with crescentic glomerulonephritis (GN) related to several mechanisms: four patients with anti-glomerular basement membrane antibodies or anti-GBM disease; 16 with immune complex mediated GN; and eight with pauci-immune GN, associated with vasculitis in four cases. A three-step immunoperoxidase technique was used on sections obtained from frozen renal biopsies. At the initial stage of evolution of the lesions, all the cells of the crescents expressed the beta 1, beta 3, alpha 1, alpha 3, and alpha V subunits of integrins, ICAM-1, and VCAM-1, and some cells expressed the alpha 2, alpha 5, alpha 6, and alpha L subunits of integrins along the plasma membrane. At a later stage, when the crescents were fibrocellular, alpha 3 and alpha 1 subunit expression was polarized, localized mainly in front of the extracellular matrix. In fibrotic crescents, the alpha 2, alpha 5, alpha 6, and alpha L chains were no longer detected, and VCAM-1 and ICAM-1 expression was decreased. VCAM-1 and ELAM-1 appeared on endothelial cells of peritubular capillaries in relation to the appearance of infiltrating inflammatory cells. The results of this study show that several adhesion molecules were expressed on cells forming crescents and were modified during crescent evolution; that these molecules were up-regulated on endothelial cells in relation to the severity of the inflammatory response; and that whatever the mechanism of the glomerulonephritis, adhesion molecule expression was identical. It can be postulated that adhesion molecules play a role in crescentic glomerulonephritis. Better knowledge of these molecules in human glomerulonephritis may open the way to a new therapeutic approach.

Cell Adhesion Molecules↗
PubMed2002

Variability of the human aryl hydrocarbon receptor nuclear translocator (ARNT) gene.

The aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in vertebrate transcriptional regulation as the common subunit of transcriptionally active complexes like the aryl hydrocarbon receptor (AHR)/ARNT heterodimer and hypoxia-inducible factor 1, mediating cellular responses to certain xenobiotics and to hypoxia, respectively. A cohort of healthy Caucasian volunteers was screened for genetic variations of ARNT. Six polymorphic sites could be identified, a variation in a G-stretch upstream of the ATG translation start site, a frequent silent mutation (G567C), two polymorphic sites in intron 9, and two single nucleotide substitutions leading to amino acid exchanges, G1531A (D511N) and T1551G (D517E). The frequencies were 0.005 for the Asn-coding allele and for the Glu-coding allele, respectively, with no linkage between these two mutations. Although no significant correlation with activities of CYP1A2, which is under regulatory control of the AHR/ARNT transcription complex, could be established, metabolic or pathological phenotypes may be associated with these variations.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed2006

A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120.

The human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein (Env) complex, a homotrimer containing gp120 surface glycoprotein and gp41 transmembrane glycoprotein subunits, mediates the binding and fusion of the virus with susceptible target cells. The Env complex is the target for neutralizing antibodies (NAbs) and is the basis for vaccines intended to induce NAbs. Early generation vaccines based on monomeric gp120 subunits did not confer protection from infection; one alternative approach is therefore to make and evaluate soluble forms of the trimeric Env complex. We have directly compared the immunogenicity in rabbits of two forms of soluble trimeric Env and monomeric gp120 based on the sequence of HIV-1(JR-FL). Both protein-only and DNA-prime, protein-boost immunization formats were evaluated, DNA-priming having little or no influence on the outcome. One form of trimeric Env was made by disrupting the gp120-gp41 cleavage site by mutagenesis (gp140(UNC)), the other contains an intramolecular disulfide bond to stabilize the cleaved gp120 and gp41 moieties (SOSIP.R6 gp140). Among the three immunogens, SOSIP.R6 gp140 most frequently elicited neutralizing antibodies against the homologous, neutralization-resistant strain, HIV-1(JR-FL). All three proteins induced NAbs against more sensitive strains, but the breadth of activity against heterologous primary isolates was limited. When antibodies able to neutralize HIV-1(JR-FL) were detected, antigen depletion studies showed they were not directed at the V3 region but were targeted at other, undefined gp120 and also non-gp120 epitopes.

AIDS Vaccines↗
PubMed1995

Transient transfection of GGH3-1' cells [GH3 cells stably transfected with the gonadotropin-releasing hormone (GnRH) receptor complementary deoxyribonucleic acid] with the carboxyl-terminal of beta-adrenergic receptor kinase 1 blocks prolactin release: evidence for a role of the G protein beta gamma-subunit complex in GnRH signal transduction.

G proteins consist of heterotrimeric alpha-, beta-, and gamma-subunits. To assess the role of the beta gamma-subunit complex in GnRH receptor-mediated signal transduction, GGH3-1' cells were transfected with plasmids PRK5-beta ARK1 (495-689) containing complementary DNA (cDNA) of the carboxyl-terminal (Gly495-Leu689) of beta-adrenergic receptor kinase 1 (beta ARK1). GGH3-1' cells are GH3 cells that have been stably transfected with rat GnRH receptor cDNA. The carboxyl region of beta ARK1 (Gly495-Leu689) binds to the beta gamma complex and thereby inhibits its action. Twenty-four hours after stimulation, PRL release, cAMP release, and inositol phosphate (IP) production were measured in these cells and in control cells transfected with vector PRK5 cDNA alone. In cells expressing the beta ARK1-(495-689) sequence there was inhibition of basal PRL release (69.3%), cAMP release (61.2%), and IP production (75.5%) compared to cells containing vector only. When cells expressing the beta ARK1 fragment were stimulated with a GnRH analog (Buserelin; 10(-7) M), maximal responses were inhibited (66.1% for PRL release, 52.3% for cAMP release, and 79.1% for IP production). Scatchard analysis of GnRH analog binding was also performed in the two groups of transfected cells. No significant differences in Kd or receptor numbers were found between beta ARK1-(495-689)-transfected cells and control cells containing the vector alone. These data suggest a role for the beta gamma complex in mediation of cAMP release, IP production, and hormone release in response to agonist occupancy of the GnRH receptor.

Animals↗
PubMed1991

A major substrate of maturation promoting factor identified as elongation factor 1 beta gamma delta in Xenopus laevis.

Protein synthesis is believed to be under control of the cell cycle during meiosis and mitosis. Any relationship between substrates for cdc2 kinase and components of the protein synthetic apparatus would therefore be of prime importance. During meiosis of Xenopus laevis oocytes one of the substrates for this kinase is a p47 protein, which is complexed to two other proteins, P36 and P30. Judged from partial amino acid sequence data on P47 and P30, the P30 and P47 proteins were reported to resemble the protein synthetic elongation factors (EF) 1 beta and 1 gamma from Artemia salina (Bellé, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R., and Mulner-Lorillon, O. (1989) FEBS Lett. 255, 101-104). This paper shows that the complex composed of P30, P47, and P36 from Xenopus is identical to the complex of EF-1 beta, EF-1 gamma, and EF-1 delta from Artemia according to two criteria. 1) Both stimulate elongation factor 1 alpha-mediated transfer RNA binding to ribosomes and exchange of guanine nucleotides on elongation factor 1 alpha to a comparable degree. 2) Each of the three subunits of the protein complex P30.P47.P36 from Xenopus shows a structural homology with one of the corresponding subunits of EF-1 beta gamma delta from Artemia. Presumably the phosphorylation of EF-1 gamma, which associates with tubulin at least in vitro, is important in processes following the onset of meiosis which is accompanied by a rise of protein synthesis.

Amino Acid Sequence↗
PubMed2005

Molecular basis for the specificity of p27 toward cyclin-dependent kinases that regulate cell division.

The cyclin-dependent kinase inhibitors (CKIs) bind to and directly regulate the catalytic activity of cyclin-dependent kinase (Cdk)/cyclin complexes involved in cell cycle control and do not regulate other, closely related Cdks. We showed previously that the CKI, p27, binds to Cdk2/cyclin A though a sequential mechanism that involves folding-on-binding. The first step in the kinetic mechanism is interaction of a small, highly dynamic domain of p27 (domain 1) with the cyclin subunit of the Cdk2/cyclin A complex, followed by much slower binding of a more lengthy and less flexible domain (domain 2) to Cdk2. The second step requires folding of domain 2 into the kinase inhibitory conformation. Rapid binding of p27 domain 1 to cyclin A tethers the inhibitor to the binary Cdk2/cyclin A complex, which reduces the entropic barrier associated with slow binding of domain 2 to the catalytic subunit. We show here that p27/cyclin interactions are an important determinant of p27 specificity towards cell cycle Cdks. We used surface plasmon resonance, limited proteolysis, mass spectrometry, and NMR spectroscopy to study the interaction of p27 with Cdk2/cyclin A, and with another Cdk complex, Cdk5/p25, that is involved in neurodegeneration. Importantly, Cdk5/p35 (the parent complex of Cdk5/p25) is not regulated by p27 in neurons. Our results show that p27 binds to Cdk5 and Cdk2 with similar, slow kinetics. However, p27 fails to interact with p25 within the Cdk5/p25 complex, which we believe prevents formation of a kinetically trapped, inhibited p27/Cdk5/p25 complex in vivo. The helical topology of p25 is very similar to that of cyclin A. However, p25 lacks the MRAIL sequence in one helix that, in the cell cycle cyclins, mediates specific interactions with domain 1 of p21 and p27. Our results strongly suggest that p21 and p27, related Cdk inhibitors, select their cell cycle regulatory Cdk targets by binding specifically to the cyclin subunit of these Cdk/cyclin complexes as a first step in a sequential, folding-on-binding mechanism.

Amino Acid Sequence↗
PubMed2005

Cutting Edge: NF-kappaB-activating kinase-associated protein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter molecule-1-mediated IFN regulatory factor 3 activation.

TLRs signal the presence of microbial patterns and activate transcription factors. In TLR3 and TLR4, the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM-1) (also called Toll/IL-1R domain-containing adapter inducing IFN-beta (TRIF)) mediates IFN regulatory factor 3 (IRF3) phosphorylation followed by IFN-beta production. The regulatory subunit TNFR-associated factor family member-associated NF-kappaB activator (TANK) couples with the kinase complex IkappaB kinase-related kinase epsilon/NF-kappaB-activating kinase (NAK) (TANK-binding kinase 1 (TBK1)) that involveTICAM-1-dependent IFN-beta induction. There are several TANK-homologous proteins. We tested whether TICAM-1 binds and coprecipitates with TANK or its family proteins. The results are: 1) the TANK family protein NAK-associated protein 1 (NAP1), but not TANK, coprecipitates withTICAM-1; 2) NAP1 overexpression markedly enhances TBK1-mediated IFN-beta promoter activation; 3) a dominant-negative form, NAP (158-270), suppresses IRF3 activation in response to poly(I:C) or LPS; 4) RNA interference targeting of the NAP1 message results in a failure of poly(I:C)-mediated IRF3 polymerization and IFN-beta production. Thus, NAP1 is the kinase subunit responsible for TLR3/4-mediated IFN-beta induction in the TICAM-1 pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1989

Human melanoma cells express a novel integrin receptor for laminin.

This study sought to determine whether human melanoma cells express integrin-related receptors that mediate their adhesion to laminin. We found that antibodies against the integrin beta 1 chain blocked cell attachment to laminin-coated surfaces. Furthermore, immunofluorescence staining demonstrated beta 1 complexes in vinculin-positive focal adhesion plaques on the basal surface of cells attached to laminin substrates. Chromatography of detergent extracts of 125I-surface-labeled cells on laminin-Sepharose columns recovered two major laminin-binding proteins (100 and 130 kDa, reduced) that bound with high affinity to the columns and were eluted with EDTA. Both proteins were specifically immunoprecipitated from column fractions with monoclonal and polyclonal antibodies to the integrin beta 1 subunit, indicating that they form a noncovalent heterodimer complex. The alpha-like subunit is composed of a 30-kDa light chain that is joined by a disulfide bond to the 100-kDa heavy chain. This complex was not recovered from columns of fibronectin- or collagen type I- or IV-Sepharose. Laminin-binding by the alpha beta 1 complex was independent of Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg-like sequences, but required the presence of divalent cations. The 100-kDa alpha-like subunit was electrophoretically and immunochemically distinct from the other known alpha subunits, alpha 1-alpha 6. The results indicate that human melanoma cells express a novel laminin-specific integrin beta 1 complex which may mediate the cells' interactions with this ligand.

Antibodies, Monoclonal↗
PubMed1995

Different domains of the AP-1 adaptor complex are required for Golgi membrane binding and clathrin recruitment.

The assembly of clathrin-coated buds on the Golgi requires the recruitment of the heterotetrameric AP-1 adaptor complex, which is dependent on both guanine nucleotides and the small GTP-binding protein ADP-ribosylation factor (ARF). Here, we have investigated the structural domains of the AP-1 complex necessary for ARF-mediated translocation of the adaptor complex onto Golgi membranes and the subsequent recruitment of clathrin onto the membrane. Controlled proteolysis of purified AP-1, derived from bovine adrenal coated vesicles, was used to generate AP-1 core fragments composed of the amino-terminal trunk regions of the beta 1 and gamma subunits and associated mu 1 and sigma 1 subunits, and lacking either the beta 1 subunit carboxyl-terminal appendage or both beta 1 and gamma subunit appendages. On addition of these truncated fragments to AP-1-depleted adrenal cytosol, both types of core fragments were efficiently recruited onto Golgi membranes in the presence of GTP gamma S. Recruitment of both core fragments was inhibited by the fungal metabolite brefeldin A, indicative of an ARF-dependent process. Limited tryptic digestion of recruited, intact cytosolic AP-1 resulted in the quantitative release of the globular carboxyl-terminal appendage domains of the beta 1 and gamma subunits. The adaptor core complex remained associated with the Golgi membranes. Recruitment of cytosolic clathrin onto the Golgi membranes was strictly dependent on the presence of intact AP-1. Tryptic removal of the beta 1 subunit appendage prevented subsequent clathrin recruitment. We conclude that the structural determinants required for the ARF-mediated binding of cytosolic AP-1 onto Golgi membranes are contained within the adaptor core, and that the carboxyl-terminal appendage domains of the beta 1 and gamma subunits do not play any role in this process. Subsequent recruitment of cytosolic clathrin, however, requires an intact beta 1 subunit.

3T3 Cells↗
PubMed1995

Zinc is a mixed antagonist of homomeric rho 1 gamma-aminobutyric acid-activated channels.

The transition metal Zn2+ is differentially distributed in the central nervous system, where it is proposed to be a neuromodulator. One of the documented effects of Zn2+ is the antagonism of gamma-aminobutyric acid (GABA)-mediated synaptic inhibition. This antagonism is presumed to result from a direct interaction of Zn2+ with the GABA receptor/ionophore complex, although the characteristics of Zn2+ sensitivity are dependent on the particular GABA subunit combination. In this study, we examined the effects of Zn2+ on homomeric rho 1 GABA-activated channels expressed in Xenopus oocytes. Zn2+ was found to be a mixed antagonist of these recombinant rho 1 GABA receptors. The antagonism was predominantly competitive at low Zn2+ concentrations (< or = 100 microM), whereas at high Zn2+ concentrations (> 100 microM) a noncompetitive antagonism was apparent. Evidence is presented showing that the antagonism was not due to an interaction of GABA and Zn2+ in solution but, rather, resulted from interactions of these two ligands with the GABA-activated channel. A mechanism is proposed for Zn(2+)-mediated antagonism in which GABA and Zn2+ bind to distinct sites on the GABA complex. The apparent mixed antagonism may arise from different Ki values for the binding of Zn2+ to non-agonist-bound or agonist-bound receptors. However, two distinct Zn2+ binding sites, one competitive and one noncompetitive, could also give rise to the dual antagonism.

Animals↗
PubMed1992

Coordinate role for cell surface chondroitin sulfate proteoglycan and alpha 4 beta 1 integrin in mediating melanoma cell adhesion to fibronectin.

Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.

Amino Acid Sequence↗
PubMed1997

Mapping of the gene encoding the integrin-linked kinase, ILK, to human chromosome 11p15.5-p15.4.

We have recently reported the identification and cloning of the gene encoding p59ILK, a novel protein ser/thr kinase that is found in physiologic complexes with beta integrin subunits. ILK is a potential protoonocogene that appears to function in mediating signal transduction by beta 1 family integrins. Fluorescence in situ hybridization analysis of metaphase and decondensed free chromatin fibers localized ILK to 11p15.5-p15.4. This position was also confirmed by relational mapping using well-characterized translocations with breakpoints in chromosome band 11p15. Our results indicate that ILK maps between HBBC and CALC loci, in the 11p15.5-p15.4 band interval. This location may be important in evaluating the potential role of p59ILK in tumorigenesis since it has been shown that this region is associated with both genomic imprinting and loss of heterozygosity in certain types of tumor.

Cell Line↗
PubMed2001

Insights into the role of the von Hippel-Lindau gene product. A key player in hypoxic regulation.

Many adaptive responses to hypoxia involve changes in gene transcription mediated by the hypoxia-inducible factor 1 complex. Central to this is oxygen-dependent proteolysis of the alpha subunit, which has recently been shown to require the von Hippel-Lindau tumour-suppressor protein. This observation provides one mechanism by which inherited defects in the von Hippel-Lindau gene could cause features of the clinical syndrome, and offers insight into the events leading to sporadic clear cell renal cancer. Furthermore, it clearly implicates the von Hippel-Lindau tumour-suppressor protein in the biochemistry of oxygen sensing.

DNA-Binding Proteins↗
PubMed1994

Selective activation of phospholipase C by recombinant G-protein alpha- and beta gamma-subunits.

Receptor activation of phospholipase C (PLC) via G-proteins occurs by pertussis toxin-sensitive and toxin-insensitive signaling pathways. The alpha-subunits of the Gq family are presumed to mediate the toxin-insensitive pathway, but the nature of the G-proteins mediating the toxin-sensitive pathway is not established. Recently, PLC-beta has been shown to be activated by G-protein beta gamma-subunits of mixed or undefined composition. The relative activities of G-protein subunits that might activate PLC-beta were examined using defined recombinant alpha- and beta gamma-subunits obtained from the baculovirus expression system by reconstituting the purified subunits with purified bovine brain PLC-beta 1 or turkey erythrocyte PLC-beta in unilamellar phospholipid vesicles. Turkey erythrocyte G alpha 11 and recombinant G alpha 11 and G alpha q obtained after expression in Sf9 cells activated both bovine brain PLC-beta 1 and turkey erythrocyte PLC-beta. In contrast, under the same assay conditions, recombinant G alpha i1, G alpha i2, G alpha i3, and G alpha o were without effect on either type of PLC. All types of beta gamma-subunits tested (r beta 1 gamma 2, r beta 1 gamma 3, r beta 2 gamma 2, r beta 2 gamma 3, bovine brain beta gamma or turkey erythrocyte beta gamma) inhibited G alpha 11-mediated activation of PLC, presumably by promotion of formation of inactive heterotrimeric G-protein. All types of beta gamma-subunits also markedly stimulated the activity of turkey erythrocyte PLC-beta but did not activate bovine brain PLC-beta 1. Of the four different beta gamma complexes of defined composition, three stimulated PLC with similar activities whereas beta 2 gamma 3 was less effective. The data suggest that pertussis toxin-sensitive activation of PLC is mediated by the beta gamma-subunits of G-proteins acting on specific phospholipase C isoenzymes.

Animals↗