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. 1998 Jun 23;95(13):7281-6.
doi: 10.1073/pnas.95.13.7281.

Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix

B E Slack  1
Affiliations

Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix

B E Slack. Proc Natl Acad Sci U S A. .

Abstract

The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal adhesion-associated proteins paxillin and focal adhesion kinase (FAK), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine-glycine-aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine-glycine-glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell's interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.

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Figures

Figure 1
Figure 1
Activation of muscarinic m3 receptors increases tyrosine phosphorylation of paxillin and FAK. (A–C) Confluent monolayers of HEK cells stably transfected with m3 receptors were treated with 100 μM carbachol or vehicle for various periods of time. (D–F) Cell monolayers were incubated for 10 min in serum-free medium containing various concentrations of carbachol. Cells were lysed, and antiphosphotyrosine immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine antibodies (A), antibodies to paxillin (B, E), or antibodies to FAK (D). (C, F) Paxillin content of antiphosphotyrosine immunoprecipitates was quantitated by densitometry. Data are expressed as a percentage of the maximal response. Means ± SEM from three to four experiments are shown.
Figure 2
Figure 2
PKC dependence of muscarinic receptor-mediated tyrosine phosphorylation. Cell monolayers were pretreated for 15 min with medium containing 2.5 μM GF 109203X or dimethyl sulfoxide (the vehicle in which the inhibitor was dissolved). The medium was replaced with fresh medium containing dimethyl sulfoxide or GF 109203X alone, or with carbachol (100 μM) or PMA (1 μM). Anti-phosphotyrosine immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine (RC20) antibodies (A), antibodies to FAK (B), or antibodies to paxillin (C). (D) Paxillin content of anti-phosphotyrosine immunoprecipitates was quantitated by densitometry. Data are expressed as a percentage of the control response. Means ± SEM from at least three experiments are shown. ∗, Significant difference from control; ∗∗, significant difference from carbachol or PMA (P < 0.05). (E) Cells in suspension were treated with 2.5 μM GF 109203X (GF109) or dimethyl sulfoxide for 15 min and then allowed to attach for 30 min to culture dishes coated with poly-d-lysine (Pdl) or fibronectin (Fn). Anti-phosphotyrosine immunoprecipitates from cell lysates were analyzed by immunoblotting with antibodies to paxillin (Upper) or FAK (Lower). Data are means ± SEM from three experiments. ∗, significant difference from control (P < 0.05).
Figure 3
Figure 3
Effect of RGD-containing peptides on muscarinic receptor- and fibronectin-mediated tyrosine phosphorylation. (A) Confluent cell monolayers were incubated for 20 min in control medium, or in medium containing carbachol (100 μM), in the presence or absence of RGDS peptide (1 mM). Cell lysates were assayed for tyrosine phosphorylation of FAK (Upper) or paxillin (Middle). The medium was assayed for soluble derivatives of the amyloid precursor protein (sAPP) (Lower). (B) Cell monolayers were treated for 20 min with or without carbachol (100 μM) in the presence or absence of GRGDS or GRGES peptides (0.75 mM) and analyzed for tyrosine phosphorylation of FAK (Upper) and paxillin (Lower). (C) Paxillin and FAK content of anti-phosphotyrosine immunoprecipitates was quantitated by densitometry. Data are expressed as relative units. Means ± SEM from three or four experiments are shown. ∗, Significant difference from corresponding control group; ∗∗, significant difference from carbachol (P < 0.05). (D) The GRGDS peptide blocked tyrosine phosphorylation stimulated by adhesion of cells for 30 min to fibronectin (Fn). No increase in tyrosine phosphorylation was observed in cells adherent to plastic (Pl) or poly-d-lysine (Pdl). (E) Cells in suspension were allowed to adhere to culture dishes coated with Pdl or Fn. After 2.5 h, cells were incubated for 20 min in fresh medium in the presence or absence of 100 μM carbachol and analyzed for tyrosine phosphorylation of FAK (Upper) and paxillin (Lower). This experiment was repeated three times with similar results.
Figure 4
Figure 4
Formation of focal contacts and stress fibers is induced by carbachol treatment. Immunofluorescence microscopy was used to visualize vinculin (A and B), a major focal adhesion-associated protein, and actin stress fibers (C and D). In serum-starved cells, vinculin staining was diffuse (A), but following exposure to 100 μM carbachol for 10 min, vinculin became concentrated in focal contacts, one of which is indicated by the arrow (B). Similarly, few stress fibers were observed in quiescent cells (C) but were a prominent feature in cells treated with carbachol (D). Actin filaments were detected with a fluorescent phalloidin conjugate. Focal adhesions were visualized with an antibody to vinculin.
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References

    1. Bonner T I, Buckley N J, Young A C, Brann M R. Science. 1987;237:527–532. - PubMed
    1. Peralta E G, Ashkenazi A, Winslow J W, Smith D H, Ramachandran J, Capon D J. EMBO J. 1987;6:3923–3929. - PMC - PubMed
    1. Bonner T I, Young A C, Brann M R, Buckley N J. Neuron. 1988;1:403–410. - PubMed
    1. Peralta E G, Ashkenazi A, Winslow J W, Ramachandran J, Capon D J. Nature (London) 1988;334:434–437. - PubMed
    1. Sandmann J, Peralta E G, Wurtman R J. J Biol Chem. 1991;266:6031–6034. - PubMed

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