Integrins are important mediators of cell adhesion to extracellular ligands and can transduce biochemical signals both into and out of cells. The cytoplasmic domains of integrins interact with several structural and signalling proteins and consequently participate in the regulation of cell shape, motility, growth and differentiation. It has been shown that calreticulin associates with the cytoplasmic domains of integrin alpha-subunits and that this interaction can influence integrin-mediated cell adhesion to extracellular matrix. We have now developed calreticulin-deficient embryonic stem (ES) cells and isolated embryonic fibroblasts from calreticulin mutant mice. We find that in both cell types integrin-mediated adhesion is severely impaired, although integrin expression is unaltered. Expression of recombinant calreticulin in double knockout ES cells by complementary DNA transfection rescued integrin-mediated adhesion. In wild-type cells, engagement of surface integrins induced a transient elevation in cytosolic calcium concentration owing to influx of extracellular calcium. This calcium transient was absent in calreticulin-deficient cells. In contrast, the amount of calcium in endomembrane stores, which is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin, was indistinguishable in the two cell types. Our results indicate that calreticulin is an essential modulator both of integrin adhesive functions and integrin-initiated signalling, but that it may not play a significant role in the storage of luminal calcium.
Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE‐1 isoform of the antiport. NHE‐1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F‐actin are concentrated at sites of NHE‐1 accumulation. A mutated construct of NHE‐1 lacking residues 566‐635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE‐1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.
Trafficking of the Na ؉ /H؉ exchanger isoform 3 (NHE3) between sub-apical vesicles and apical membrane of epithelial cells is a suggested mechanism of regulation of NHE3 activity. When epitope-tagged NHE3 was stably expressed in NHE-deficient Chinese hamster ovary cells, a sizable fraction was found in recycling endosomes. This system was used to analyze the mechanism of endocytosis of NHE3. Immunofluorescence and radiolabeling experiments showed that inhibition of clathrinmediated endocytosis using hypertonicity, acid treatment, or K ؉ depletion inhibited internalization of NHE3. Moreover, transient transfection of an inhibitory mutant of dynamin (DynS45N) blocked the clathrin-mediated uptake of transferrin, as well as the endocytosis of NHE3. In ileal villus cells, endogenous NHE3 was also found to co-purify with isolated clathrin-coated vesicles, thereby confirming their association in native tissues. The role of COP-I subunits in the intracellular traffic of NHE3 was evaluated using ldlF cells, which bear a temperature-sensitive mutation in the ⑀-COP subunit. At the permissive temperature, NHE3 distributed normally, whereas at the restrictive temperature, which induces rapid degradation of ⑀-COP, NHE3 was still internalized, but its subcellular distribution was altered. These results indicate that endocytosis of NHE3 occurs primarily via clathrin-coated pits and vesicles and that normal intracellular trafficking of NHE3 involves an ⑀-COP-dependent step.
Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M 2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M 2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M 2 receptor to enhanced green fluorescent protein-M 2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n ؍ 2, 0.495 ؎ 0.019; n ؍ 4, 0.202 ؎ 0.010; n ؍ 6, 0.128 ؎ 0.006; n ؍ 8, 0.093 ؎ 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 -0.24, identifying the M 2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.Much evidence now indicates that G protein-coupled receptors can exist as oligomers (1, 2), a development that has implications for all aspects of GPCR 4 -mediated signaling. Among the many questions prompted by the emergence of such structures is that of oligomeric size. Although commonly referred to as dimers, oligomers of GPCRs have been detected most often by means of coimmunoprecipitation or resonance energy transfer (3). As typically applied, neither technique can distinguish dimers from larger oligomers. The latter have been identified on the basis of their electrophoretic mobility (reviewed in Ref. 4), but the composition of the bands may be unclear, and the size under the conditions of electrophoresis may have little in common with that in the membrane. Larger oligomers also have been identified by approaches in which detection requires the colocalization of three or four proteins, each bearing a different tag (5-11), but such procedures place only a lower limit on the possible size of the array.There have been comparatively few attempts to examine the oligomeric status of a GPCR in a more quantitative and explicit manner. Measurements of BRET at different ratios of acceptor to donor have pointed to dimers of the melatonin receptor (12), the  1 -and  2 -adrenergic receptors (13), the M 1 , M 2 , and M 3 muscarinic receptors (14), and the neurotensin receptor (15)....
Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H ؉ transport mechanisms. We therefore investigated whether Na ؉ /H ؉ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na ؉ gradient, because the phagosomal membrane was found to be devoid of Na ؉ /K ؉ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H ؉ -ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.Leukocytes internalize invading pathogens into a membrane-bound organelle called the phagosome. The microbes are initially encircled by extensions of the plasmalemma, and the resulting nascent phagosome subsequently matures upon fusion with endosomes, lysosomes, and possibly other vesicular compartments (1-3). Acidification of the phagosomal interior is a critical component of the microbicidal response; not only is the low pH directly toxic to many microorganisms but, in addition, lytic enzymes secreted into the phagosomal lumen function optimally at acidic pH. Phagosomal acidification has been attributed principally to the activity of vacuolar type H ϩ -ATPases (V-ATPases), 1 which have been shown to accumulate in the phagosomal membrane as it matures within the cell (1, 4). The importance of V-ATPase-mediated acidification to microbial elimination is highlighted by the failure of macrophages to kill organisms such as Mycobacterium tuberculosis. This and other species of mycobacteria avoid acid-mediated degradation by preventing insertion of H ϩ pumps into the phagosomal membrane (5, 6). Interestingly, the growth of mycobacteria ceases at pH Ͻ6.2 (7), suggesting that their ability to modulate phagosomal pH is an important determinant o...
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