Abstract
The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαs and PKA.
Highlights
Secreted ligands of the Hedgehog (Hh) family function as morphogens and pattern tissues, such as the spinal cord, limb bud, and paraxial mesoderm, during development
We propose that target-cell responses to Hh signaling are influenced through both GPR161-dependent and -independent pathways that are regulated by G protein–coupled receptor kinase 2 (GRK2) and that converge on GαS and protein kinase A (PKA) activity
GPR161 functions as an attenuator of Hh signaling in NIH/3T3 cells
Summary
Secreted ligands of the Hedgehog (Hh) family function as morphogens and pattern tissues, such as the spinal cord, limb bud, and paraxial mesoderm, during development. Activation of the Hh signaling pathway in responsive cells can drive the patterning of spinal neural progenitor subtypes in a manner that depends on both the concentration of the ligand Sonic Hedgehog (SHH) and the duration of SHH exposure [1]. Patched (PTCH) proteins, the transmembrane receptors for Hh ligands, repress the activity of Smoothened (SMO), a Frizzled-family G protein–coupled receptor (GPCR) that transmits the Hh signal across the membrane to the cytoplasm. In the absence of Hh ligands, protein kinase A (PKA) and Suppressor of Fused (SUFU) inhibit the activity of the gliomaassociated oncogene family transcription factors GLI2 and GLI3 and promote the proteolysis of GLI3 into a transcriptional repressor fragment (hereafter called GLI3R) [2]. Hh ligands inactivate PTCH1, allowing SMO to adopt an active conformation and accumulate in the membrane of the primary cilium [3]. The formation of GLI3R is blocked and full-length GLI2 and GLI3 are converted to transcriptional activators (hereafter GLI2A and GLI3A) [4,5,6,7]
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