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1.
bioRxiv ; 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37214942

RESUMO

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

2.
Methods Mol Biol ; 2374: 175-184, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34562252

RESUMO

Communication between PATCHED1 (PTCH1) and SMOOTHENED (SMO) is fundamental to Hedgehog (Hh) signal transduction in development and disease. We describe a real-time cell-based SMO functional assay based on SMO activity-dependent changes in cellular cAMP concentrations. This assay is capable of detecting changes in SMO conformation within minutes of PTCH1 inactivation by Hh ligands. As a result, it expands the range of experimental perturbations that can be used to dissect PTCH1-SMO communication, enabling a deeper mechanistic understanding of a longstanding mystery in Hh signal transduction.


Assuntos
Receptor Smoothened/metabolismo , AMP Cíclico , Proteínas Hedgehog , Ligantes , Receptores Acoplados a Proteínas G , Transdução de Sinais
3.
PLoS Biol ; 19(4): e3001191, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33886552

RESUMO

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Receptor Smoothened/fisiologia , Animais , Animais Geneticamente Modificados , Domínio Catalítico/genética , Células Cultivadas , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Embrião não Mamífero , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , Transdução de Sinais/genética , Receptor Smoothened/metabolismo , Peixe-Zebra
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