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V Gray

Publications and source records attributed to V Gray.

9 recordsLinked to original sources
PubMed1992

The receptor for the basement membrane glycoprotein entactin is the integrin alpha 3/beta 1.

Entactin is a glycoprotein found in basement membranes in complex with laminin, and purified entactin can promote the attachment and spreading of cells. We report here the isolation and identification of the plasma membrane receptor for entactin from PC-3 human prostate carcinoma cells which attach and spread on entactin. The receptor was isolated by affinity chromatography on mouse recombinant entactin-Sepharose of 125I surface-labeled octyl glucoside cell extracts. The receptor, which consisted of two polypeptides of relative molecular masses of 150 and 116 kDa, bound to the entactin-Sepharose matrix in the presence of CaCl2, MgCl2, and MnCl2, and was eluted with EDTA, but not with Arg-Gly-Asp-containing peptides. Utilizing anti-integrin antibodies, the heterodimeric receptor was identified as the integrin alpha 3 beta 1. Purified alpha 3 beta 1 bound to entactin Sepharose in a divalent cation-dependent manner and liposomes prepared with fractions eluted from the entactin-Sepharose matrix, as well as purified alpha 3 beta 1 also bound to entactin. Liposomes prepared with other integrins such as alpha 2 beta 1 did not bind to entactin. Antibody inhibition assays demonstrated that an anti-alpha 3 antibody (P1B5) inhibited the attachment of PC-3 cells to entactin whereas this antibody did not inhibit the attachment of these cells to laminin. Attachment to laminin was, however, blocked by anti-alpha 6 antibody (G0H3). These data demonstrate that the cell surface receptor for entactin on these prostate carcinoma cells is the integrin alpha 3 beta 1 and that these cells utilize alpha 6 beta 1 as the receptor for laminin.

Animals↗
PubMed1992

Identification and characterization of a novel high-molecular-weight form of the integrin alpha 3 subunit.

The integrin alpha 3 beta 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti-alpha 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected alpha 3 beta 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa alpha 3 and 115-kDa beta 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-alpha 3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa alpha 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa alpha 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa alpha 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the alpha 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa alpha 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human alpha 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin alpha 3 subunit consisting of the alpha 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative "light" chain to the 25-kDa light chain of the alpha 3 subunit.

Amino Acid Sequence↗
PubMed1991

Induction of expression of the alpha v beta 1 and alpha v beta 3 integrin heterodimers during retinoic acid-induced neuronal differentiation of murine embryonal carcinoma cells.

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process.

Amino Acid Sequence↗
PubMed1991

In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin alpha subunits.

We endeavored to identify proteins interacting with KLGFFKR, a highly conserved motif in the cytoplasmic domain adjacent to the transmembrane domain of the alpha subunit of integrins. We found that affinity chromatography of cell extracts with this peptide followed by elution with EDTA resulted in the isolation of a 60-kDa protein (p60). The N-terminal amino acid sequence of this 60-kDa polypeptide was found to be highly homologous to the Ro/SS-A antigen, a 60-kDa protein homologous to calreticulin and Aplysia "memory molecule". The binding of p60 was found to be specific for the KLGFFKR sequence since this polypeptide did not bind to a peptide with a scrambled amino acid sequence (KLRFGFK), and it was also specifically eluted from the KLGFFKR affinity matrix ith soluble KLGFFKR peptide but not with the scrambled peptide. Solid phase in vitro binding assays demonstrated specific interaction of p60 with integrin alpha 3 and alpha 5 subunits but not with the beta 1 subunit. Furthermore, p60 could be copurified with alpha 3 beta 1 following coincubation in vitro. These interactions could be inhibited by KLGFFKR peptide and also by EDTA, indicating sequence-specific and divalent cation dependent binding. Despite the fact that calreticulin is thought to be localized in the endoplasmic reticulum, a pool of Ro/SS A antigen homologous 60-kDa polypeptide was found to be present in the soluble cytoplasm, indicating the feasibility of an interaction of p60 with the integrin alpha subunits. Our data suggest that p60 (Ro/SS-A Ag) can specifically bind to integrin alpha subunits via the highly conserved KLGFFKR amino acid sequence.

Amino Acid Sequence↗
PubMed1991

Regulation of phosphoenolpyruvate carboxylase from maize leaves by nitrate and alanine.

Nitrate and alanine were found to stimulate partially purified maize leaf phosphoenolpyruvate carboxylase under specific assay conditions. Both metabolites stimulated the enzyme at low pH (7.0-7.5) and low substrate levels (1mM phosphoenolpyruvate). Nitrate was found to have a biphasic effect on the enzyme, stimulating at low concentrations (1mM-3mM), with a decrease in stimulation at higher levels. Nitrate caused inhibition of activity at pH 8.0 and although alanine caused some stimulation in activity at pH 8.0 this was not as marked as at the lower pH levels.

Alanine↗
PubMed1990

Blood-borne collagenous debris complexes with plasma fibronectin after thermal injury.

Plasma fibronectin augments the clearance of blood-borne foreign and effete complexes by mononuclear phagocytes. The release of a "gelatin-like" ligand into plasma after thermal injury has been reported. We quantified the release of this collagenous debris from thermally injured skin, and its potential interaction with soluble fibronectin in plasma using anesthetized rats. Collagen-like material debris in the plasma was detected by assay of hydroxyproline. Fibronectin was measured by a double antibody enzyme-linked immunosorbent assay (ELISA) technique. Over a 24-hour postburn interval, plasma hydroxyproline increased from 6.7 +/- 0.6 micrograms/mL to a maximum of 19.0 +/- 3.3 micrograms/mL at 60 minutes postburn, and normalized by 6 hours. A direct correlation existed between the magnitude of burn injury and the increase in plasma hydroxyproline. In parallel, plasma fibronectin declined over a 15-minute to 2-hour period postburn, and normalized by 3 to 4 hours with rebound hyperfibronectinemia observed at 24 hours. The elevation in total plasma hydroxyproline was not due to an increase in plasma Clq (zero time, 26.2 +/- 1.4 micrograms/mL; 60 minutes, 23.9 +/- 1.1 micrograms/mL). Tracer studies with 125I-fibronectin showed that the acute decline of plasma fibronectin was due to its uptake by the liver and binding to sites of tissue injury. Total hydroxyproline in extracts of burn skin, used as an index of soluble collagenous material, rose from 15 +/- 3.3 micrograms/g skin at zero time to 129.3 +/- 43.7 micrograms/g skin by 5 minutes postburn, with a decline to 38 +/- 22 micrograms/g skin by 24 hours. The formation of circulating fibronectin-gelatin complexes in vivo was documented by cross-immunoelectrophoresis coupled with autoradiography using 125I-gelatin as a model ligand. Thus, collagenous tissue debris from burned skin may enter the plasma after thermal injury and directly complexes with soluble fibronectin before hepatic phagocytic clearance.

Animals↗
PubMed1990

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v.

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.

Cell Adhesion↗
PubMed1977

Reticuloendothelial phagocytic response to bacterial challenge after traumatic shock.

Resistance to intravenous (IV) and intraperitoneal (IP) bacterial challenge during periods of reticuloendothelial (RE) depression following trauma as well as the influence of bacteremia on RE phagocytosis were studied. The experimental shock model utilized was the anesthetized (2 mg/100 g sodium pentobarbital) male rat subjected to nonlethal Noble-Collip drum trauma. During post-traumatic RE depression (60 min after injury) rats were challenged IV or IP with Escherichia coli (1.02 X 10(10)). The clearance half-time of the bacterial load injected intravenously in controls was 1.23 +/- 0.10 min. In contrast, the half-time was 3.62 +/- 0.69 min after sublethal trauma (p less than 0.005) and associated with prolonged blood bacterial retention. Pulmonary localization of E. coli administered either IV or IP was elevated in traumatized rats. Comparison of routes of bacterial challenge with respect to blood levels of viable bacteria suggested lower host bacterial resistance to the IP injection as opposed to the IV route of administration. Production of experimental bacteremia in normal rats resulted in a 39% depression (p less than 0.01) of RE test colloid clearance rate accompanied by a 49% increase (p less than 0.01) in pulmonary colloid localization. The data suggest that depressed systemic RE clearance capacity following trauma may decrease systemic resistance to septicemia, and that severe bacteremia may further undermine the functional state of the reticuloendothelial system.

Animals↗