Signal transduction in primary cilia – analyzing and manipulating GPCR and second messenger signaling

AbstractThe second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted in distinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist, and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

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The influence of angiotensin II and cyclic nucleotide second messenger signal transduction on ischemia-reperfusion-induced elevations in microvascular hydraulic permeability

Journal of Surgical Research ◽

10.1016/j.jss.2005.11.185 ◽

2006 ◽

Vol 130 (2) ◽

pp. 227

Author(s):

R. Ramirez ◽

J. Sadjadi ◽

B. Curran ◽

G.P. Victorino

Keyword(s):

Signal Transduction ◽

Angiotensin Ii ◽

Cyclic Nucleotide ◽

Ischemia Reperfusion ◽

Second Messenger ◽

Hydraulic Permeability

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Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816614115 ◽

2018 ◽

Vol 115 (49) ◽

pp. E11465-E11474 ◽

Cited By ~ 15

Author(s):

F. Donelson Smith ◽

Mitchell H. Omar ◽

Patrick J. Nygren ◽

Joseph Soughayer ◽

Naoto Hoshi ◽

...

Keyword(s):

Solid Phase ◽

Second Messenger ◽

Nuclear Accumulation ◽

Nucleotide Polymorphisms ◽

Binding Assays ◽

Subcellular Targeting ◽

Regulatory Subunits ◽

Anchoring Proteins ◽

Anchoring Protein ◽

Second Messenger Signaling

A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca2+ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

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CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids

10.1101/2021.03.15.435490 ◽

2021 ◽

Author(s):

Megan Lo ◽

Amnon Sharir ◽

Michael D Paul ◽

Hayarpi Torosyan ◽

Christopher Agnew ◽

...

Keyword(s):

Signal Transduction ◽

Primary Cilia ◽

Molecular Mechanisms ◽

Negative Regulator ◽

Membrane Composition ◽

Hh Signaling ◽

Pathway Regulation ◽

Patched 1 ◽

Cancer Signal Transduction

The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can cause congenital malformations, such as digit anomalies and holoprosencephaly, and other diseases, including cancer. Signal transduction is initiated by HH ligand binding to the Patched 1 (PTCH1) receptor on primary cilia, thereby releasing inhibition of Smoothened (SMO), a HH pathway activator. Although cholesterol and several oxysterol lipids, which are enriched in the ciliary membrane, play a crucial role in HH activation, the molecular mechanisms governing the regulation of these lipid molecules remain unresolved. Here, we identify Canopy 4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that controls membrane sterol lipid levels. Cnpy4—/— embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway at the level of SMO in vitro, and elevates membrane levels of accessible sterol lipids such as cholesterol, an endogenous ligand involved in SMO activation. Thus, our data demonstrate that CNPY4 is a negative regulator that fine-tunes the initial steps of HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.

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