Limits...
Integrin-specific signaling pathways controlling focal adhesion formation and cell migration.

Mostafavi-Pour Z, Askari JA, Parkinson SJ, Parker PJ, Ng TT, Humphries MJ - J. Cell Biol. (2003)

Bottom Line: After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties.First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not.Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

Show MeSH

Related in: MedlinePlus

Characterization of recombinant H/120-GAG-ABC. (A) ELISA assay showing the binding of HepII (9E9, 16E6, 36C9, 39B6, 26G10, and 19B7), IIICS-B (18D8) and IIICS-C (10G6) mAbs to immobilized H/120 (solid bars) and its heparin-binding deficient mutant H/120-GAG-ABC (open bars), each coated at 10 μg/ml. (B) Attachment of A375-SM melanoma cells to native H/120 (closed diamonds) and H/120-GAG-ABC (open circles). The level of nonspecific binding, determined from A375-SM melanoma cell attachment to wells coated with BSA alone, was subtracted. Values shown in both panels are mean ± SD of triplicate wells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172880&req=5

fig3: Characterization of recombinant H/120-GAG-ABC. (A) ELISA assay showing the binding of HepII (9E9, 16E6, 36C9, 39B6, 26G10, and 19B7), IIICS-B (18D8) and IIICS-C (10G6) mAbs to immobilized H/120 (solid bars) and its heparin-binding deficient mutant H/120-GAG-ABC (open bars), each coated at 10 μg/ml. (B) Attachment of A375-SM melanoma cells to native H/120 (closed diamonds) and H/120-GAG-ABC (open circles). The level of nonspecific binding, determined from A375-SM melanoma cell attachment to wells coated with BSA alone, was subtracted. Values shown in both panels are mean ± SD of triplicate wells.

Mentions: To assess folding of H/120-GAG-ABC, an ELISA assay was performed in which the binding of a panel of HepII/IIICS mAbs was determined. Some of these mAbs, including 9E9 and 16E6, do not Western blot, and therefore recognize conformational epitopes within FN. As shown in Fig. 3 A, all mAbs recognized H/120-GAG-ABC to a similar extent to native H/120 with the exception of 9E9, where binding was reduced by 50%. It is possible that one or more arginine or lysine residues contribute to the epitope for this mAb. The binding of biotinylated heparin to the mutant protein was then investigated. The level of binding to H/120-GAG-ABC was reduced by >98% compared with that of native H/120, and was not significantly different from the background, indicating that all key heparin-binding sites had been removed (unpublished data). Recombinant native and mutated proteins were then tested for their ability to support A375-SM melanoma cell adhesion. The proteins supported attachment in a dose-dependent manner (Fig. 3 B). Attachment to native H/120 variant was maximal at a level of 80%, and a coating concentration of ∼0.7 μg/ml was required for half-maximal attachment. H/120-GAG-ABC showed only slightly lower activity, with a maximal level of cell attachment of ∼65% and a coating concentration of 1.6 μg/ml being required for half-maximal attachment. Finally, H/120-GAG-ABC was unable to trigger vinculin recruitment to focal adhesions when A375-SM cells were prespread on 50K (unpublished data).


Integrin-specific signaling pathways controlling focal adhesion formation and cell migration.

Mostafavi-Pour Z, Askari JA, Parkinson SJ, Parker PJ, Ng TT, Humphries MJ - J. Cell Biol. (2003)

Characterization of recombinant H/120-GAG-ABC. (A) ELISA assay showing the binding of HepII (9E9, 16E6, 36C9, 39B6, 26G10, and 19B7), IIICS-B (18D8) and IIICS-C (10G6) mAbs to immobilized H/120 (solid bars) and its heparin-binding deficient mutant H/120-GAG-ABC (open bars), each coated at 10 μg/ml. (B) Attachment of A375-SM melanoma cells to native H/120 (closed diamonds) and H/120-GAG-ABC (open circles). The level of nonspecific binding, determined from A375-SM melanoma cell attachment to wells coated with BSA alone, was subtracted. Values shown in both panels are mean ± SD of triplicate wells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172880&req=5

fig3: Characterization of recombinant H/120-GAG-ABC. (A) ELISA assay showing the binding of HepII (9E9, 16E6, 36C9, 39B6, 26G10, and 19B7), IIICS-B (18D8) and IIICS-C (10G6) mAbs to immobilized H/120 (solid bars) and its heparin-binding deficient mutant H/120-GAG-ABC (open bars), each coated at 10 μg/ml. (B) Attachment of A375-SM melanoma cells to native H/120 (closed diamonds) and H/120-GAG-ABC (open circles). The level of nonspecific binding, determined from A375-SM melanoma cell attachment to wells coated with BSA alone, was subtracted. Values shown in both panels are mean ± SD of triplicate wells.
Mentions: To assess folding of H/120-GAG-ABC, an ELISA assay was performed in which the binding of a panel of HepII/IIICS mAbs was determined. Some of these mAbs, including 9E9 and 16E6, do not Western blot, and therefore recognize conformational epitopes within FN. As shown in Fig. 3 A, all mAbs recognized H/120-GAG-ABC to a similar extent to native H/120 with the exception of 9E9, where binding was reduced by 50%. It is possible that one or more arginine or lysine residues contribute to the epitope for this mAb. The binding of biotinylated heparin to the mutant protein was then investigated. The level of binding to H/120-GAG-ABC was reduced by >98% compared with that of native H/120, and was not significantly different from the background, indicating that all key heparin-binding sites had been removed (unpublished data). Recombinant native and mutated proteins were then tested for their ability to support A375-SM melanoma cell adhesion. The proteins supported attachment in a dose-dependent manner (Fig. 3 B). Attachment to native H/120 variant was maximal at a level of 80%, and a coating concentration of ∼0.7 μg/ml was required for half-maximal attachment. H/120-GAG-ABC showed only slightly lower activity, with a maximal level of cell attachment of ∼65% and a coating concentration of 1.6 μg/ml being required for half-maximal attachment. Finally, H/120-GAG-ABC was unable to trigger vinculin recruitment to focal adhesions when A375-SM cells were prespread on 50K (unpublished data).

Bottom Line: After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties.First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not.Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

Show MeSH
Related in: MedlinePlus