PubMed · 11960205
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John M Pettifor. 2002-04-19. Rickets.. https://doi.org/10.1007/s00223-001-0046-x
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Platelet adhesion on von Willebrand factor (vWf) requires the co-ordinated adhesive function of glycoprotein Ib/V/IX and integrin alphaIIbbeta3. Recent evidence [Nesbitt, Kulkarni, Giuliano, Gonclaves, Dopheide, Yap, Harper, Salem and Jackson (2002) J. Biol. Chem. 277, 2965-2972] suggests that outside-in signals from both receptors play important roles in regulating platelet-adhesion dynamics under flow. In the present study, we have examined the mechanisms utilized by protein kinase C (PKC) to promote irreversible platelet adhesion on vWf. We demonstrate that PKC is primarily activated downstream of integrin alphaIIbbeta3, not glycoprotein Ib, during platelet adhesion on vWf. This integrin alphaIIbbeta3-dependent PKC activation establishes a positive-feedback loop that promotes further integrin alphaIIbbeta3 activation, calcium mobilization and firm platelet adhesion. This feedback loop appears to be most relevant at relatively low cytosolic calcium concentrations (mean Delta[Ca(2+)](i);100 nM) as artificially elevating calcium (mean Delta[Ca(2+)](i) > 500 nM) induces integrin alphaIIbbeta3 activation and irreversible platelet adhesion independent of PKC. Our studies demonstrate the existence of a complex signalling relationship operating between PKC, cytosolic calcium and integrin alphaIIbbeta3 that serves to regulate platelet-adhesion dynamics under flow. Furthermore, we have established the existence of PKC-dependent and -independent pathways regulating integrin alphaIIbbeta3 activation and stable platelet adhesion on vWf.
Each of three internal domains of multi-modular cellobiohydrolase CbhA from Clostridium thermocellum, X1(1), X1(2) (previously designated as fibronectin type 3-like modules, Fn3(1) and Fn3(2)) and family 3 carbohydrate-binding module (CBM3) binds 1 mol of Ca(2+). Structures and thermal stabilities of X1(1), X1(2), CBM3, X1(1)X1(2), and X1(1)X1(2)-CBM3 containing Ca(2+) (holo-proteins) and without Ca(2+) (apo-proteins) have been studied using CD spectroscopy. All domains are beta-proteins with irregular far-UV CD spectra due to the aromatic side chain contributions. The positive signal at 294 nm in the near-UV CD spectrum of X1(1) lacking a tryptophan residue might be attributed to the presence of aromatic clusters. Thermal denaturation of all proteins is reversible and results in the total loss of tertiary structure and preservation of significant amount of ordered secondary structure. Removal of Ca(2+) destabilizes polypeptides in a different way and to a different extent. It decreases the melting temperature ( T (m)) (by 20 degrees C) and co-operativity of thermal transition of X1(1), increases the number of transitions and lowers the co-operativity of unfolding of CBM3, and slightly decreases T (m)s (2.4-4.2 degrees C) of X1(2), X1(1)X1(2), and X1(1)X1(2)-CBM3. Transitions of X1(1)X1(2) and X1(1)X1(2)-CBM3 follow a two-state model regardless of the presence of Ca(2+). X1(1) is strongly stabilized in the apo-X1(1)X1(2) and apo-X1(1)X1(2)-CBM3 as they display T (m)s similar to those of individual and combined holo-modules. Observed CD spectra of X1(1)X1(2) and X1(1)X1(2)-CBM3 differ from those calculated as the simple weighted sum of individual modules. These differences are more prominent in spectra of apo-proteins. The results indicate the presence of inter-domain interactions in CbhA. Holo-modules, i.e. containing Ca(2+), behave essentially independently, but in the absence of Ca(2+) domain interactions are more important for the conformation of the polypeptides.
We previously reported that activation of M(3) muscarinic acetylcholine receptors (mAChR) generates anti-proliferative signals and stimulates cadherin-mediated adhesion in the SCC-9 small cell lung carcinoma (SCLC) cell line. The current study was undertaken to determine the frequency of functional mAChR expression among different SCLC cell lines, and to test the ability of mAChR to generate anti-proliferative signals in different SCLC cell lines. The potential role of Rac1 in SCLC cell-cell adhesion was also investigated. Exposure to the mAChR agonist carbachol induces robust Ca(2+) mobilization (indicated by intracellular fluorescence of the Ca(2+)-binding dye Indo-1) in three SCLC cell lines (SCC-9, SCC-15, and NCI-H146), modest Ca(2+) mobilization in one SCLC cell line (NCI-H209), and no detectable Ca(2+) mobilization in two SCLC cell lines (SCC-18 and NCI-H82). The M(3) mAChR-selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibits Ca(2+) mobilization in all SCLC cell lines responding to carbachol. Incubation with carbachol for four hours significantly inhibits [3H]thymidine uptake in three of the four SCLC cell lines expressing functional mAChR (SCC-9, SCC-15, and NCI-H146 cells), but does not significantly alter [3H]thymidine uptake in the other SCLC cell lines examined. These results indicate that SCLC cell lines often express functional mAChR which elicit anti-proliferative signals when activated. To investigate the role of Rac1 in SCLC adhesion, SCC-9 cells were transiently transfected with cDNA constructs coding for Rac1, constitutively active Rac1(Val-12), or dominant negative Rac1(Asn-17) tagged to green fluorescent protein (GFP). SCC-9 cells expressing GFP-tagged constitutively active Rac1(Val-12) exhibit increased cell-cell adhesion in comparison to cells expressing GFP-Rac1 or GFP-Rac1(Asn-17). Constitutively active GFP-Rac1(Val-12), but not GFP-Rac1 or GFP-Rac1(Asn-17), accumulates at cell-cell junctions in SCC-9 cells. These results indicate that activated Rac1 increases SCLC cell-cell adhesion, consistent with the possibility that Rac1 activation contributes to increased SCLC cell-cell adhesion induced by mAChR stimulation. These findings indicate that activation of mAChR may play a significant role in regulating the proliferation and adhesion of SCLC cells. The demonstration by other investigators that acetylcholine is expressed by a variety of cells in the airways supports the possibility that acetylcholine may activate mAChR expressed by SCLC cells in primary tumors.