The signaling achieved by the Hedgehog family of secreted proteins plays an essential role in vertebrate development and maintenance of several postembryonic stem cell populations. An obligatory component of the pathway utilized by Hedgehogs is Smoothened, a 7- transmembrane (7-TM) protein. Smoothened couples unequivocally to all members of the Gi family of heterotrimeric G proteins. While Gi can be required in the activation of Gli transcription factors (Gli), a premise of this proposal is that G provides to Hedgehogs, through 'noncanonical' signaling, the ability to expand well beyond the bounds of Gli in their actions. An additional premise is that Gli can be controlled through 7-TM receptors apart from Smoothened.
The first aim i s to evaluate determinants of a tension we believe to exist between canonical and noncanonical signaling. We suspect, and will test, that Smo's sequestration into the primary cilium and/or arrestin's actions apart from facilitating sequestration occurs at the expense of noncanonical signaling. Indices of the latter, i.e. Gi activity, are the activation of RhoA and inhibition of adenylyl cyclase. We will also evaluate in this aim the nature of a permissive interface, in certain cells, that exists between Gi and canonical signaling.
The second aim i s to evaluate differential engagement of canonical and noncanonical signaling by ligands. We will evaluate the role of Gi in establishing Smo's affinity for agonists, the possibility that canonical and noncanonical signaling are sequentially rather than simultaneously engaged as a function of agonist concentration, and the likelihood of biased agonism. The underpinnings of this work are assays of radiolabel displacement, arrestin recruitment, gli1 and RhoA activation, and Smo translocation.
The third aim i s to evaluate the nature and consequences of activation of Gli by G proteins apart from Gi, with a focus on G13. Of interest in this regard are the intrinsic activities of G? subunits, the mechanism by which G13 controls Gli, and the similarity of G13 to Hedgehogs in the nature and extent of this control.

Public Health Relevance

The Hedgehogs are proteins that play an essential role in embryonic development and tissue regeneration. An understanding of the mechanisms by which these proteins signal or by which the signaling can be replicated will provide important leads into derangements in pathological contexts and consequent therapeutic manipulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM080396-08
Application #
9187832
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Melillo, Amanda A
Project Start
2007-09-24
Project End
2018-11-30
Budget Start
2016-12-01
Budget End
2018-11-30
Support Year
8
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Pharmacology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Ho Wei, Lan; Arastoo, Mohammad; Georgiou, Ioanna et al. (2018) Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia. PLoS One 13:e0203170
Cheng, Lan; Al-Owais, Moza; Covarrubias, Manuel L et al. (2018) Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K+ currents in cardiomyocytes and prolongs cardiac action potential duration. J Biol Chem 293:11022-11032
Guo, Xueshui; Riobo-Del Galdo, Natalia A; Kim, Eun Ji et al. (2018) Overlap in signaling between Smoothened and the ? subunit of the heterotrimeric G protein G13. PLoS One 13:e0197442
Manning, David R; Shen, Feng; Riobo, Natalia A (2015) Evaluating the Activity of Smoothened Toward G Proteins Using [³?S]Guanosine 5'-(3-O-thio)triphosphate ([³?S]GTP?S). Methods Mol Biol 1322:35-44
Shen, Feng; Cheng, Lan; Douglas, Andrew E et al. (2013) Smoothened is a fully competent activator of the heterotrimeric G protein G(i). Mol Pharmacol 83:691-7
Douglas, Andrew E; Heim, Jennifer A; Shen, Feng et al. (2011) The alpha subunit of the G protein G13 regulates activity of one or more Gli transcription factors independently of smoothened. J Biol Chem 286:30714-22
Polizio, Ariel H; Chinchilla, Pilar; Chen, Xiaolen et al. (2011) Sonic Hedgehog activates the GTPases Rac1 and RhoA in a Gli-independent manner through coupling of smoothened to Gi proteins. Sci Signal 4:pt7
Polizio, Ariel H; Chinchilla, Pilar; Chen, Xiaole et al. (2011) Heterotrimeric Gi proteins link Hedgehog signaling to activation of Rho small GTPases to promote fibroblast migration. J Biol Chem 286:19589-96