Differences between high- and low-motility buffalo sperm identified by comparative proteomics.

This study was undertaken to investigate differences in protein expression between high- and low-motility sperm of swamp buffalo. The research used two-dimensional gel electrophoresis (2DE) coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS) to analyse the different proteins. The results showed 18 different expression protein spots between high- and low-motility buffalo sperm; eight of these proteins were up-regulated in low-motility sperm, five were down-regulated, one deleted and four proteins specifically expressed. Finally, four proteins were successfully identified by MS as belonging to three unique proteins; they are outer dense fibre of sperm tails protein 2 (ODF2), ATP synthase subunit alpha (ATP5A1) and succinyl-CoA synthetase subunit beta (SUCLG2). In summary, these results help to develop an understanding of the molecular mechanisms associated with low-motility sperm and provide clues for finding molecular markers associated with sperm motility.

Mechanisms of Ganweikang Tablets against Chronic Hepatitis B: A Comprehensive Study of Network Analysis, Molecular Docking, and Chemical Profiling.

The hepatitis B virus (HBV) is one of the major viral infection problems worldwide in public health. The exclusive proprietary Chinese medicine Ganweikang (GWK) tablet has been marketed for years in the treatment of chronic hepatitis B (CHB). However, the pharmacodynamic material basis and underlying mechanism of GWK are not completely clear. This study is aimed at investigating the pharmacological mechanism of the GWK tablet in the treatment of CHB. The chemical ingredient information was obtained from the Traditional Chinese Medicine Database and Analysis Platform (TCMSP), Traditional Chinese Medicines Integrated Database (TCMID), and Shanghai Institute of Organic Chemistry of CAS. Ingredients and disease-related targets were defined by a combination of differentially expressed genes from CHB transcriptome data and open-source databases. Target-pathway-target (TPT) network analysis, molecular docking, and chemical composition analysis were adopted to further verify the key targets and corresponding active ingredients of GWK. Eight herbs of GWK were correlated to 330 compounds with positive oral bioavailability, and 199 correlated targets were identified. The TPT network was constructed based on the 146 enriched targets by KEGG pathway analysis, significantly associated with 95 pathways. Twenty-five nonvolatile components and 25 volatile components in GWK were identified in UPLC-QTOF/MS and GC-MS chromatograms. The key active ingredients of GWK include ferulic acid, oleanolic acid, ursolic acid, tormentic acid, 11-deoxyglycyrrhetic acid, dibenzoyl methane, anisaldehyde, wogonin, protocatechuic acid, psoralen, caffeate, dimethylcaffeic acid, vanillin, ß-amyrenyl acetate, formonentin, aristololactam IIIa, and 7-methoxy-2-methyl isoflavone, associated with targets CA2, NFKB1, RELA, AKT1, JUN, CA1, CA6, IKBKG, FOS, EP300, CREB1, STAT1, MMP9, CDK2, ABCB1, and ABCG2.

Vitamin D receptor gene BsmI polymorphism B allele, but not BB genotype, is associated with systemic lupus erythematosus in a Han Chinese population.

The vitamin D receptor (VDR) gene is a candidate gene for susceptibility to autoimmune disorders. Association studies of VDR polymorphisms and risk of systemic lupus erythematosus (SLE) have often produced conflicting results in different ethnic backgrounds. The aim of this study is to test the association between VDR gene BsmI polymorphism and the genetic susceptibility to SLE in a Han Chinese population. Three hundred and thirty-seven patients with SLE and 239 healthy controls were genotyped for the VDR gene BsmI polymorphism (rs1544410) by polymerase chain reaction and restriction fragment length polymorphism analysis in this study, after which the relationship between BsmI polymorphisms and the mRNA expression of VDR, as well as clinical manifestations in patients with SLE, was evaluated. It was found that the frequency of B allele was significantly increased in SLE relative to the control group (χ(2) = 4.681, p = 0.031), although the distribution of VDR BsmI polymorphism genotype frequencies did not differ significantly between patients and controls (χ(2) = 4.098, p = 0.129). Moreover, VDR B allele was found to be associated with lupus nephritis (p = 0.027) and also with production of anti-nucleosome antibodies (p = 0.037). The mRNA of VDR was markedly down-regulated in patients with VDR B allele compared with that in patients without B allele (p = 0.016). Our results indicate a possible role of genetic factors (the VDR B allele) in influencing disease susceptibility in Han Chinese patients. Also, VDR B allele is associated with the development of nephritis and the down-regulation of VDR mRNA expression in SLE.

Regulation of the PH-domain-containing tyrosine kinase Etk by focal adhesion kinase through the FERM domain.

Etk/BMX, a member of the Btk family of tyrosine kinases, is highly expressed in cells with great migratory potential, including endothelial cells and metastatic carcinoma cell lines. Here, we present evidence that Etk is involved in integrin signalling and promotes cell migration. The activation of Etk by extracellular matrix proteins is regulated by FAK through an interaction between the PH domain of Etk and the FERM domain of FAK. The lack of Etk activation by extracellular matrix in FAK-null cells could be restored by co-transfection with wild-type FAK. Disrupting the interaction between Etk and FAK diminished the cell migration promoted by either kinase. Furthermore, inhibiting Etk expression in metastatic carcinoma cell lines with an antisense oligonucleotide blocks integrin-mediated migration of these cells. Taken together, our data indicate the essential role of the interaction of the PH domain of Etk and the FERM domain of FAK in integrin signalling.

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-alpha4 beta1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130 cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.

Regulation of the cell cycle by focal adhesion kinase.

In this report, we have analyzed the potential role and mechanisms of integrin signaling through FAK in cell cycle regulation by using tetracycline-regulated expression of exogenous FAK and mutants. We have found that overexpression of wild-type FAK accelerated G1 to S phase transition. Conversely, overexpression of a dominant-negative FAK mutant DeltaC14 inhibited cell cycle progression at G1 phase and this inhibition required the Y397 in DeltaC14. Biochemical analyses indicated that FAK mutant DeltaC14 was mislocalized and functioned as a dominant-negative mutant by competing with endogenous FAK in focal contacts for binding signaling molecules such as Src and Fyn, resulting in a decreases of Erk activation in cell adhesion. Consistent with this, we also observed inhibition of BrdU incorporation and Erk activation by FAK Y397F mutant and FRNK, but not FRNKDeltaC14, in transient transfection assays using primary human foreskin fibroblasts. Finally, we also found that DeltaC14 blocked cyclin D1 upregulation and induced p21 expression, while wild-type FAK increased cyclin D1 expression and decreased p21 expression. Taken together, these results have identified FAK and its associated signaling pathways as a mediator of the cell cycle regulation by integrins.

Suppression of Pyk2 kinase and cellular activities by FIP200.

Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase-interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200-Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.

Retroviral expression of alternatively spliced forms of rat fibronectin.

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.

Expression of normal and mutant avian integrin subunits in rodent cells.

We describe the expression of the beta 1 subunit of avian integrin in rodent cells with the purpose of examining the structure-function relationships of various domains within this subunit. The exogenous subunit is efficiently and stably expressed in 3T3 cells, and it forms hybrid heterodimers with endogenous murine alpha subunits, including alpha 3 and alpha 5. These heterodimers are exported to the cell surface and localize in focal contacts where both extracellular matrix and cytoskeleton associate with the plasma membrane. Hybrid heterodimers consisting of exogenous beta 1 and endogenous alpha subunits bind effectively and specifically to columns of cell-binding fragments of fibronectin. The exogenous avian beta 1 subunit appears to function as well as its endogenous murine equivalent, consistent with the high degree of conservation noted previously for integrins. In contrast, expression of a mutant form of avian integrin beta 1 subunit lacking the cytoplasmic domain produces hybrid heterodimers which, while efficiently exported to the cell surface and still capable of binding fibronectin, do not localize efficiently in focal contacts. This further implicates the cytoplasmic domain of the beta 1 subunit in interactions required for cytoskeletal organization.

Identification of p130Cas as a mediator of focal adhesion kinase-promoted cell migration.

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130(Cas), two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130(Cas) association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130(Cas) and its subsequent binding to several SH2 domains, which depended on both the p130(Cas) binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130(Cas) further increased cell migration on FN and coexpression of the p130(Cas) SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130(Cas), but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130(Cas) complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.

Lack of association between HLA-G 14-bp polymorphism and systemic lupus erythematosus in a Han Chinese population.

HLA-G is a non-classical HLA-class Ib molecule with multiple immunoregulatory properties. A 14-bp insertion/deletion polymorphism in the HLA-G gene has been suggested to influence the expression of HLA-G and to associate with certain pathological conditions, including autoimmune diseases. We investigated the influence of the 14-bp insertion/deletion polymorphism in the HLA-G gene on disease susceptibility in systemic lupus erythematosus by genotyping this polymorphism in 231 patients with systemic lupus erythematosus and 367 healthy controls and analyzing the levels of soluble HLA-G in a subset of patients with systemic lupus erythematosus and healthy subjects from a Han Chinese population. No statistically significant differences were observed in the frequencies of the 14-bp insertion/deletion HLA-G alleles or genotypes between controls and patients with systemic lupus erythematosus. However, a significant increased expression of soluble HLA-G was noted in patients with systemic lupus erythematosus (mean value = 230.2 U/ml vs 118.3 U/ml in controls, p = 0.0001). Moreover, patients with high levels of soluble HLA-G presented with higher disease activity and had more neurological involvement. Our results do not support the HLA-G 14-bp insertion/deletion polymorphism as a genetic factor influencing systemic lupus erythematosus susceptibility. It is possible that the expression of soluble HLA-G in systemic lupus erythematosus is enhanced as part of a mechanism to try to restore the tolerance process towards auto-antigens and to counteract inflammation. However, the participation of this molecule in the pathological process of the disease also could not be excluded.

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.

Transcriptional activation of cyclin D1 promoter by FAK contributes to cell cycle progression.

Integrin-mediated cell adhesion to the extracellular matrix is required for normal cell growth. Cyclin D1 is a key regulator of G1-to-S phase progression of the cell cycle. Our previous studies have demonstrated that integrin signaling through focal adhesion kinase (FAK) plays a role in the regulation of cell cycle progression, which correlates with changes in the expression of cyclin D1 and the cdk inhibitor, p21, induced by FAK. In this report, we first investigated the roles of both cyclin D1 and p21 in the regulation of cell cycle progression by FAK. We found that overexpression of a dominant-negative FAK mutant DeltaC14 suppressed cell cycle progression in p21(-/-) cells as effectively as in the control p21(+/+) cells. Furthermore, we found that overexpression of ectopic cyclin D1 could rescue cell cycle inhibition by DeltaC14. These results suggested that cyclin D1, but not p21, was the primary functional target of FAK signaling pathways in cell cycle regulation. We then investigated the mechanisms underlying the regulation of cyclin D1 expression by FAK signaling. Using Northern blotting and cyclin D1 promoter/luciferase assays, we showed that FAK signaling regulated cyclin D1 expression at the transcriptional level. Using a series of cyclin D1 promoter mutants in luciferase assays as well as electrophoretic mobility shift assays (EMSA), we showed that the EtsB binding site mediated cyclin D1 promoter regulation by FAK. Finally, we showed that FAK regulation of cyclin D1 depends on integrin-mediated cell adhesion and is likely through its activation of the Erk signaling pathway. Together, these studies demonstrate that transcriptional regulation of cyclin D1 by FAK signaling pathways contributes to the regulation of cell cycle progression in cell adhesion.

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas.

Metaplastic breast carcinoma is an aggressive form of invasive breast cancer with histological evidence of epithelial to mesenchymal transition (EMT). However, the defining molecular events are unknown. Here we show that CCN6 (WISP3), a secreted matricellular protein of the CCN (CYR61/CTGF/NOV) family, is significantly downregulated in clinical samples of human spindle cell metaplastic breast carcinoma. We generated a mouse model of mammary epithelial-specific Ccn6 deletion by developing a floxed Ccn6 mouse which was bred with an MMTV-Cre mouse. Ccn6fl/fl;MMTV-Cre mice displayed severe defects in ductal branching and abnormal age-related involution compared to littermate controls. Ccn6fl/fl;MMTV-Cre mice developed invasive high grade mammary carcinomas with bona fide EMT, histologically similar to human metaplastic breast carcinomas. Global gene expression profiling of Ccn6fl/fl mammary carcinomas and comparison of orthologous genes with a human metaplastic carcinoma signature revealed a significant overlap of 87 genes (P=5 × 10-11). Among the shared deregulated genes between mouse and human are important regulators of epithelial morphogenesis including Cdh1, Ck19, Cldn3 and 4, Ddr1, and Wnt10a. These results document a causal role for Ccn6 deletion in the pathogenesis of metaplastic carcinomas with histological and molecular similarities with human disease. We provide a platform to study new targets in the diagnosis and treatment of human metaplastic carcinomas, and a new disease relevant model in which to test new treatment strategies.

Galanin-like peptide promotes feeding behaviour via activation of orexinergic neurones in the rat lateral hypothalamus.

Galanin-like peptide (GALP) is produced in neurones in the hypothalamic arcuate nucleus and is implicated in the neural control of feeding behaviour. Previously, we have reported that GALP immunoreactive fibres were in direct contact with orexin/hypocretin immunoreactive neurones in the rat lateral hypothalamus using double-immunofluorescence. Centrally administered GALP is known to stimulate feeding behaviour. However, the target neurones of this action have not been clarified. The present study aimed to determine features of the GALP-mediated neuronal feeding pathway in rat. Accordingly, at the ultrastructural level, GALP-immunoreactive axon terminals were found to make synapses on orexin/hypocretin immunoreactive cell bodies and dendritic processes in the lateral hypothalamus. c-Fos immunoreactivity was expressed in orexin/hypocretin-immunoreactive neurones but not in melanin concentrating hormone-immunoreactive neurones in the lateral hypothalamus at 90 min after the application of GALP by i.c.v. infusion. Furthermore, to determine whether GALP regulates feeding behaviour via orexin/hypocretin neurones, the feeding behaviour of rats was studied following GALP i.c.v. injection with or without anti-orexin A and B immunoglobulin (IgG) pretreatment. The anti-orexin IgGs markedly inhibited GALP-induced hyperphagia. These results suggest that orexin/hypocretin-containing neurones in the lateral hypothalamus are targeted by GALP, and that GALP-induced hyperphagia is mediated via orexin/hypocretin neurones in the rat hypothalamus.

Identification of a novel interaction between integrin beta1 and 14-3-3beta.

Integrins are cell surface receptors for extracellular matrix, which play important roles in a variety of biological processes. 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in regulation of various cellular functions. Here, we report identification of an interaction between the integrin beta1 cytoplasmic domain and 14-3-3beta by using the yeast two-hybrid screen. Like several other proteins, the integrin beta1 cytoplasmic domain associated with 14-3-3beta by a non-phosphoserine mechanism. The 14-3-3beta/integrin beta1 interaction was confirmed by in vitro binding assays as well as co-precipitation in vivo. Furthermore, we found that 14-3-3beta co-localized with integrin beta1 during the early stage of cell spreading on fibronectin, suggesting a potential role of the 14-3-3beta/integrin beta1 interaction in the regulation of cell adhesion. Using tetracycline-regulated expression system, we showed that overexpression of 14-3-3beta stimulated cell spreading and migration on fibronectin but not on poly-L-lysine. However, the induced expression of 14-3-3beta did not affect tyrosine phosphorylation of FAK or its substrates, p130(cas) and paxillin, suggesting that 14-3-3beta regulated integrin-mediated cell spreading and migration by FAK-independent mechanisms. Taken together, these results identify an interaction between integrin and 14-3-3 proteins and suggest a potentially novel cellular function for 14-3-3 proteins in the regulation of integrin-mediated cell adhesion and signaling events.

The Grb7 family proteins: structure, interactions with other signaling molecules and potential cellular functions.

Grb7 family adaptor molecules consist of Grb7, Grb10 and Grb14, each of which has several splicing variants. Like other adaptor molecules, Grb7 family proteins function to mediate the coupling of multiple cell surface receptors to downstream signaling pathways in the regulation of various cellular functions. They share significant sequence homology with each other and a conserved molecular architecture including an amino-terminal proline-rich region, a central segment termed the GM region (for Grb and Mig) which includes a PH domain and shares sequence homology with the Caenorhabditis elegans protein, Mig-10, involved in embryonic migration, and a carboxyl-terminal SH2 domain. Grb7 family proteins are differentially expressed in a variety of tissues. They are phosphorylated on serine/threonine as well as tyrosine residues, although the kinases responsible have not been well characterized. Grb7 family proteins are mainly localized in the cytoplasm, but have been observed at the plasma membrane, focal contacts, or mitochondria under certain conditions. A large number of receptor tyrosine kinases and other signaling molecules can associate with Grb7 family proteins, mostly through the SH2 domains. Various isoforms of Grb10 have been shown to regulate cell proliferation and apoptosis, whereas Grb7 has been found to regulate cell migration and also implicated in tumor progression. Future studies of interests will include identification of potential downstream effectors of Grb7 family proteins as well as understanding of the mechanisms of specificity of the different family members in signal transduction.

Signal transduction in cell-matrix interactions.

Cell-matrix interactions play critical roles in a variety of physiological and pathological processes by regulating cell migration, proliferation, and differentiation. Integrin family cell-adhesion receptors have been identified as the major receptors for various extracellular matrix proteins and shown to be capable of transducing biochemical signals across the plasma membrane to regulate cellular functions. Protein tyrosine phosphorylation has been implicated in a central role in integrin-initiated signal transduction. Other signaling pathways have also been shown to be activated by cell-matrix interactions. This chapter reviews our current understanding of the key molecular components and their interactions in the integrin-signaling pathways and examines the role of various signaling pathways and molecular players in the context of cellular functions regulated by cell-matrix interactions.