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Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration.

ABSTRACT: Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a G(i)-coupled receptor contrasts with the lack of an apparently consistent requirement for G(i) in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-G(i) coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for G(i) in Shh-induced fibroblast migration. We found that Shh acutely stimulates the small Rho GTPases Rac1 and RhoA via SMO through a G(i) protein- and PI3K-dependent mechanism, and that these are required for cell migration. These responses were independent of transcription by Gli and of the C-terminal domain of SMO, as we show using a combination of molecular and genetic tools. Our findings provide a mechanistic model for fibroblast migration in response to Shh and underscore the role of SMO-G(i) coupling in non-canonical Hh signaling.

SUBMITTER: Polizio AH

PROVIDER: S-EPMC3103338 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration.

Polizio Ariel H AH Chinchilla Pilar P Chen Xiaole X Kim Sangbumn S Manning David R DR Riobo Natalia A NA

The Journal of biological chemistry 20110407 22

Evidence supporting the functionality of Smoothened (SMO), an essential transducer in most pathways engaged by Hedgehog (Hh), as a G(i)-coupled receptor contrasts with the lack of an apparently consistent requirement for G(i) in Hh signal transduction. In the present study, we sought to evaluate the role of SMO-G(i) coupling in fibroblast migration induced by Sonic Hedgehog (Shh). Our results demonstrate an absolute requirement for G(i) in Shh-induced fibroblast migration. We found that Shh acut ...[more]

PMID: 21474452

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Project description:BackgroundPrevious research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking.MethodsWe conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics.ResultsWe demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS.ConclusionsSRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.

Dataset Information

Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration.

Publications

Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration.

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