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Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

Spicer E, Suckert C, Al-Attar H, Marsden M - PLoS ONE (2010)

Bottom Line: We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling.Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells.These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.

ABSTRACT
During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

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Kermit2 interacts with the cytoplasmic domain of the α5 and α6 integrin subunits.(A) Yeast two two-hybrid assays were conducted using α5, α6 and αV cytoplasmic domains as bait in combination with kermit2 or kermit2mut as prey. The data is presented as average normalized β-galactosidase activity (±SD). The interaction of kermit2 with α5 and α6 is abolished by the AEEL mutation in kermit2mut (* P<0.002). The αV subunit does not interact with kermit2 or kermit2mut (** indicates P<0.001 between α5 or α6 and αV bait constructs). (B) GST pulldowns. HA tagged Kermit2 is detected in lysates (lane 1 input), and is pulled down with a GST-α5 fusion construct (lane 2 GST-α5). Most of the kermit2 remains in the supernatant (lane 3 sup). A control GST-β1 construct (lane 5 GST-β1) does not pull Kermit2 from lysates (lane 4 input, lane 6 sup). (C) Coimmunoprecipitation of kermit2 with α5β1 integrin. HA tagged kermit2 is detected in lysates (lane 1 input). Kermit2 is found in association with immunoprecipitated α5β1 (lane 2 P8D4), while a significant portion of kermit2 remains in the supernatant (sup).
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pone-0010665-g001: Kermit2 interacts with the cytoplasmic domain of the α5 and α6 integrin subunits.(A) Yeast two two-hybrid assays were conducted using α5, α6 and αV cytoplasmic domains as bait in combination with kermit2 or kermit2mut as prey. The data is presented as average normalized β-galactosidase activity (±SD). The interaction of kermit2 with α5 and α6 is abolished by the AEEL mutation in kermit2mut (* P<0.002). The αV subunit does not interact with kermit2 or kermit2mut (** indicates P<0.001 between α5 or α6 and αV bait constructs). (B) GST pulldowns. HA tagged Kermit2 is detected in lysates (lane 1 input), and is pulled down with a GST-α5 fusion construct (lane 2 GST-α5). Most of the kermit2 remains in the supernatant (lane 3 sup). A control GST-β1 construct (lane 5 GST-β1) does not pull Kermit2 from lysates (lane 4 input, lane 6 sup). (C) Coimmunoprecipitation of kermit2 with α5β1 integrin. HA tagged kermit2 is detected in lysates (lane 1 input). Kermit2 is found in association with immunoprecipitated α5β1 (lane 2 P8D4), while a significant portion of kermit2 remains in the supernatant (sup).

Mentions: A robust interaction between kermit2 and the α5 and α6 subunits was observed in two hybrid assays. This interaction is abolished when the central PDZ domain of kermit2 is mutated from ALGL to AEEL (kermit2mut). As expected there was no observable interaction between kermit2 and the αV cytoplasmic domain (Figure 1A). The interaction between the α6 cytoplasmic domain is stronger than that with the α5 cytoplasmic domain and likely reflects the differences between the SEA and SDA motif as steady state expression levels of both bait and prey constructs are similar in all transformants (Figure S1). As the α6 subunit is not expressed during gastrulation we concentrated our efforts on further characterizing the α5 subunit interaction.


Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

Spicer E, Suckert C, Al-Attar H, Marsden M - PLoS ONE (2010)

Kermit2 interacts with the cytoplasmic domain of the α5 and α6 integrin subunits.(A) Yeast two two-hybrid assays were conducted using α5, α6 and αV cytoplasmic domains as bait in combination with kermit2 or kermit2mut as prey. The data is presented as average normalized β-galactosidase activity (±SD). The interaction of kermit2 with α5 and α6 is abolished by the AEEL mutation in kermit2mut (* P<0.002). The αV subunit does not interact with kermit2 or kermit2mut (** indicates P<0.001 between α5 or α6 and αV bait constructs). (B) GST pulldowns. HA tagged Kermit2 is detected in lysates (lane 1 input), and is pulled down with a GST-α5 fusion construct (lane 2 GST-α5). Most of the kermit2 remains in the supernatant (lane 3 sup). A control GST-β1 construct (lane 5 GST-β1) does not pull Kermit2 from lysates (lane 4 input, lane 6 sup). (C) Coimmunoprecipitation of kermit2 with α5β1 integrin. HA tagged kermit2 is detected in lysates (lane 1 input). Kermit2 is found in association with immunoprecipitated α5β1 (lane 2 P8D4), while a significant portion of kermit2 remains in the supernatant (sup).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871791&req=5

pone-0010665-g001: Kermit2 interacts with the cytoplasmic domain of the α5 and α6 integrin subunits.(A) Yeast two two-hybrid assays were conducted using α5, α6 and αV cytoplasmic domains as bait in combination with kermit2 or kermit2mut as prey. The data is presented as average normalized β-galactosidase activity (±SD). The interaction of kermit2 with α5 and α6 is abolished by the AEEL mutation in kermit2mut (* P<0.002). The αV subunit does not interact with kermit2 or kermit2mut (** indicates P<0.001 between α5 or α6 and αV bait constructs). (B) GST pulldowns. HA tagged Kermit2 is detected in lysates (lane 1 input), and is pulled down with a GST-α5 fusion construct (lane 2 GST-α5). Most of the kermit2 remains in the supernatant (lane 3 sup). A control GST-β1 construct (lane 5 GST-β1) does not pull Kermit2 from lysates (lane 4 input, lane 6 sup). (C) Coimmunoprecipitation of kermit2 with α5β1 integrin. HA tagged kermit2 is detected in lysates (lane 1 input). Kermit2 is found in association with immunoprecipitated α5β1 (lane 2 P8D4), while a significant portion of kermit2 remains in the supernatant (sup).
Mentions: A robust interaction between kermit2 and the α5 and α6 subunits was observed in two hybrid assays. This interaction is abolished when the central PDZ domain of kermit2 is mutated from ALGL to AEEL (kermit2mut). As expected there was no observable interaction between kermit2 and the αV cytoplasmic domain (Figure 1A). The interaction between the α6 cytoplasmic domain is stronger than that with the α5 cytoplasmic domain and likely reflects the differences between the SEA and SDA motif as steady state expression levels of both bait and prey constructs are similar in all transformants (Figure S1). As the α6 subunit is not expressed during gastrulation we concentrated our efforts on further characterizing the α5 subunit interaction.

Bottom Line: We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling.Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells.These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.

ABSTRACT
During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

Show MeSH
Related in: MedlinePlus