Fibroblast growth factor (FGF) homologous factors share structural but not functional homology with FGFs

Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1-FHF4; also known as FGF11-FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-A resolution. The FHF1b core domain assumes a beta-trefoil fold consisting of 12 antiparallel beta strands (beta 1 through beta 12). The FHF1b beta-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the beta 4-beta 5 loop and Val95 in the beta 9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets.     

Analysis of cadherin/catenin complexes in transformed thyroid epithelial cells: modulation by beta 1 integrin subunit

We have analysed the expression of cadherin/catenin complex molecules in PC C13 rat thyroid cells transformed in vitro with different oncogenes. No significant downregulation of either E-cadherin, alpha-, beta- and gamma-catenin was detected following the introduction of activated forms of myc, adenovirus E1A, ras, raf, myc + ras, E1A + raf. However, ras- and raf-transformed PC C13 cells showed altered adherens junctions. An altered distribution of cadherin/catenin complexes characterized by radially oriented membrane spikes perpendicular to cell edges was the most prominent feature evidenced by immunofluorescence. No beta1 integrin localization was observed in areas where this altered pattern of E-cadherin expression was detected. However, beta1 integrin subunit expression was detected at areas of cell-cell contact where E-cadherin showed a normal pattern of expression. Furthermore, ras- and raf-transformed PC C13 cells showed the ability to migrate in collagen gels, in contrast to their normal untransformed counterpart. Overexpression of beta1 integrin was found to restore normal E-cadherin localization at cell-cell contacts and to partially inhibit the ability to migrate in collagen gels. Finally, two cell lines obtained by ras transformation in vivo, and derived from a rat primary thyroid carcinoma (TK6) and its lung metastasis (MPTK6), were found to have lost gamma-catenin expression. TK6 lost also E-cadherin expression and membrane localization of alpha-catenin. These results suggest that: i) in vitro thyroid cell transformation is associated to a change in cadherin/catenin complexes distribution rather than to a decrease in expression; ii) in vivo transformation is associated to the loss of expression of some of these molecules likely due to tumor progression; iii) alterations in beta1 integrin subunit expression can result in changes in cadherin/catenin function thus implying that an integrin-cadherin synergy may exist in thyroid cells.     

Diurnal opposite variation between angiotensinase activities in photo–neuro–endocrine tissues of rats

Central and peripheral renin–angiotensin systems (RASs) act in a coordinated manner for the physiologic functions regulated by neuroendocrine events. However, whereas the diurnal rhythm of peripheral circulatory and tissue RASs is well known, the circadian behaviour of their components in central photo–neuro–endocrine structures, key elements for the control of circadian rhythms, has been barely studied. In the present study, we analysed the aspartyl- (AspAP) and glutamyl-aminopeptidase (GluAP) (aminopeptidase A) activities, the angiotensinases responsible for the metabolism of Ang I to Ang 2–10 and Ang II to Ang III, respectively, in the retina, anterior hypothalamus and pituitary at different light and dark time-points of a 12:12 h light:dark cycle (7–19 h light), using arylamide derivatives as substrates. The results demonstrated that while retina and pituitary exhibited their highest levels of AspAP activity in the light period and the lowest in the dark one, the contrary occurred in the hypothalamus – the lowest levels were observed in light conditions and the highest in darkness. The outcome for GluAP showed the highest levels in the light period and the lowest in the dark one in the three tissues analysed. In conclusion, changes in angiotensinase activities throughout the daytime may cause changes of their respective substrates and derived peptides and, consequently, in their functions. This observation may have implications for the treatment of hypertension.     

Mitochondrial localization unveils a novel role for GRK2 in organelle biogenesis

Metabolic stimuli such as insulin and insulin like growth factor cause cellular accumulation of G protein coupled receptor kinase 2 (GRK2), which in turn is able to induce insulin resistance. Here we show that in fibroblasts, GRK2 is able to increase ATP cellular content by enhancing mitochondrial biogenesis; also, it antagonizes ATP loss after hypoxia/reperfusion. Interestingly, GRK2 is able to localize in the mitochondrial outer membrane, possibly through one region within the RGS homology domain and one region within the catalytic domain. In vivo, GRK2 removal from the skeletal muscle results in reduced ATP production and impaired tolerance to ischemia. Our data show a novel sub-cellular localization of GRK2 in the mitochondria and an unexpected role in regulating mitochondrial biogenesis and ATP generation.     

Protein-tyrosine phosphatase PTPD1 regulates focal adhesion kinase autophosphorylation and cell migration

PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1(C1108S)) or the FERM domain (PTPD1(Delta1-325)) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.     

NK- and CD8(+) T cell-mediated eradication of established tumors by peritumoral injection of CpG-containing oligodeoxynucleotides.

Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODN) are known to act as adjuvants and powerful activators of the innate immune system. We investigated the therapeutic effect of CpG-ODN on a variety of established mouse tumors including AG104A, IE7 fibrosarcoma, B16 melanoma, and 3LL lung carcinoma. These tumors are only weakly immunogenic and notoriously difficult to treat. Repeated peritumoral injection of CpG-ODN resulted in complete rejection or strong inhibition of tumor growth, whereas systemic application had only partial effects. The CpG-ODN-induced tumor rejection was found to be mediated by both NK and tumor-specific CD8(+) T cells. Comparison of parental tumors and variants rendered more antigenic by transfection with tumor Ags suggested that the efficiency of the CpG-ODN therapy correlated with the antigenicity of the tumors. Peritumoral CpG-ODN treatment was even effective in a situation where the immune system was tolerant for the tumor Ag, as shown by breakage of tolerance and tumor elimination. These results suggest that peritumoral application of CpG-ODN acts locally by inducing NK cells, and also leads to efficient presentation of tumor Ags and stimulation of CD8(+) effector and memory T cells, thus providing a powerful antitumor therapy that can be also applied without knowledge of the tumor Ag.