ABSTRACT
Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.
Subject(s)
Connexins , Gap Junctions , Patch-Clamp Techniques , Humans , HEK293 Cells , Connexins/genetics , Connexins/metabolism , Gap Junctions/metabolism , Gap Junctions/genetics , Connexin 43/genetics , Connexin 43/metabolism , CRISPR-Cas Systems , Genetic Engineering/methods , Gene Knockout Techniques/methodsABSTRACT
Dysfunctional paracrine signaling through Pannexin 1 (PANX1) channels is linked to several adult neurological pathologies and emerging evidence suggests that PANX1 plays an important role in human brain development. It remains unclear how early PANX1 influences brain development, or how loss of PANX1 alters the developing human brain. Using a cerebral organoid model of early human brain development, we find that PANX1 is expressed at all stages of organoid development from neural induction through to neuroepithelial expansion and maturation. Interestingly, PANX1 cellular distribution and subcellular localization changes dramatically throughout cerebral organoid development. During neural induction, PANX1 becomes concentrated at the apical membrane domain of neural rosettes where it co-localizes with several apical membrane adhesion molecules. During neuroepithelial expansion, PANX1-/- organoids are significantly smaller than control and exhibit significant gene expression changes related to cell adhesion, WNT signaling and non-coding RNAs. As cerebral organoids mature, PANX1 expression is significantly upregulated and is primarily localized to neuronal populations outside of the ventricular-like zones. Ultimately, PANX1 protein can be detected in all layers of a 21-22 post conception week human fetal cerebral cortex. Together, these results show that PANX1 is dynamically expressed by numerous cell types throughout embryonic and early fetal stages of human corticogenesis and loss of PANX1 compromises neuroepithelial expansion due to dysregulation of cell-cell and cell-matrix adhesion, perturbed intracellular signaling, and changes to gene regulation.
ABSTRACT
The human lens is an avascular organ, and its transparency is dependent on gap junction (GJ)-mediated microcirculation. Lens GJs are composed of three connexins with Cx46 and Cx50 being expressed in lens fiber cells and Cx43 and Cx50 in the epithelial cells. Impairment of GJ communication by either Cx46 or Cx50 mutations has been shown to be one of the main molecular mechanisms of congenital cataracts in mutant carrier families. The docking compatibility and formation of functional heterotypic GJs for human lens connexins have not been studied. Previous study on rodent lens connexins revealed that Cx46 can form functional heterotypic GJs with Cx50 and Cx43, but Cx50 cannot form heterotypic GJ with Cx43 due to its second extracellular (EL2) domain. To study human lens connexin docking and formation of functional heterotypic GJs, we developed a genetically engineered HEK293 cell line with endogenously expressed Cx43 and Cx45 ablated. The human lens connexins showed docking compatibility identical to those found in the rodent connexins. To reveal the structural mechanisms of the docking incompatibility between Cx50 and Cx43, we designed eight variants based on the differences between the EL2 of Cx50 and Cx46. We found that Cx50I177L is sufficient to establish heterotypic docking with Cx43 with some interesting gating properties. Our structure models indicate this residue is important for interdomain interactions within a single connexin, Cx50 I177L showed an increased interdomain interaction which might alter the docking interface structure to be compatible with Cx43.NEW & NOTEWORTHY The human lens is an avascular organ, and its transparency is partially dependent on gap junction (GJ) network composed of Cx46, Cx50, and Cx43. We found that human Cx46 can dock and form functional heterotypic GJs with Cx50 and Cx43, but Cx50 is unable to form functional heterotypic GJs with Cx43. Through mutagenesis and patch-clamp study of several designed variants, we found that Cx50 I177L was sufficient to form functional heterotypic GJs with Cx43.
Subject(s)
Connexin 43 , Lens, Crystalline , Humans , Connexin 43/genetics , Connexin 43/metabolism , HEK293 Cells , Gap Junctions/metabolism , Connexins/genetics , Connexins/metabolism , Ion Channels/metabolism , Lens, Crystalline/metabolismABSTRACT
Every single cell in the body communicates with nearby cells to locally organize activities with their neighbors and dysfunctional cell-cell communication can be detrimental during cell lineage commitment, tissue patterning and organ development. Pannexin channels (PANX1, PANX2, and PANX3) facilitate purinergic paracrine signaling through the passage of messenger molecules out of cells. PANX1 is widely expressed throughout the body and has recently been identified in human oocytes as well as 2 and 4-cell stage human embryos. Given its abundance across multiple adult tissues and its expression at the earliest stages of human development, we sought to understand whether PANX1 impacts human induced pluripotent stem cells (iPSCs) or plays a role in cell fate decisions. Western blot, immunofluorescence and flow cytometry reveal that PANX1 is expressed in iPSCs as well as all three germ lineages derived from these cells: ectoderm, endoderm, and mesoderm. PANX1 demonstrates differential glycosylation patterns and subcellular localization across the germ lineages. Using CRISPR-Cas9 gene ablation, we find that loss of PANX1 has no obvious impact on iPSC morphology, survival, or pluripotency gene expression. However, PANX1 gene knockout iPSCs exhibit apparent lineage specification bias under 3-dimensional spontaneous differentiation into the three germ lineages. Indeed, loss of PANX1 increases representation of endodermal and mesodermal populations in PANX1 knockout cells. Importantly, PANX1 knockout iPSCs are fully capable of differentiating toward each specific lineage when exposed to the appropriate external signaling pressures, suggesting that although PANX1 influences germ lineage specification, it is not essential to this process.
ABSTRACT
During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate direct cell-cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (GJA1) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our GJA1-/- iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.
Subject(s)
CRISPR-Cas Systems , Cell Lineage , Connexin 43/deficiency , Gene Deletion , Germ Layers/metabolism , Induced Pluripotent Stem Cells/metabolism , Connexin 43/metabolism , Female , HumansABSTRACT
When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.
Subject(s)
Connexin 43/physiology , Connexins/physiology , Keratinocytes/physiology , Animals , Animals, Newborn , Cell Differentiation/genetics , Cells, Cultured , Connexin 43/genetics , Connexins/genetics , Gap Junctions/genetics , Gap Junctions/metabolism , Gene Knockdown Techniques , Mice , Mice, Inbred C57BL , Rats , Rodentia , Skin/cytology , Skin/metabolismABSTRACT
GJB2 gene (that encodes Cx26) mutations are causal of hearing loss highlighting the importance of Cx26-based channel signaling amongst the supporting cells in the organ of Corti. While the majority of these GJB2 mutations are inherited in an autosomal recessive manner, others are inherited in an autosomal dominant manner and lead to syndromic hearing loss as well as skin diseases. To assess if common or divergent mechanisms are at the root of GJB2-linked hearing loss, we expressed several mutants in cochlear-relevant HEI-OC1 cells derived from the developing organ of Corti. Since supporting cells of the mature mammalian organ of Corti have negligible Cx43, but HEI-OC1 cells are rich in Cx43, we first used CRISPR-Cas9 to ablate endogenous Cx43, thus establishing a connexin-deficient platform for controlled reintroduction of hearing-relevant connexins and Cx26 mutants. We found three distinct outcomes and cellular phenotypes when hearing loss-linked Cx26 mutants were expressed in cochlear-relevant cells. The dominant syndromic Cx26 mutant N54K had trafficking defects and did not fully prevent wild-type Cx26 gap junction plaque formation but surprisingly formed gap junctions when co-expressed with Cx30. In contrast, the dominant syndromic S183F mutant formed gap junctions incapable of transferring dye and, as expected, co-localized in the same gap junctions as wild-type Cx26 and Cx30, but also gained the capacity to intermix with Cx43 within gap junctions. Both recessive non-syndromic Cx26 mutants (R32H and R184P) were retained in intracellular vesicles including early endosomes and did not co-localize with Cx30. As might be predicted, none of the Cx26 mutants prevented Cx43 gap junction plaque formation in Cx43-rich HEI-OC1 cells while Cx43-ablation had little effect on the expression of reference genes linked to auditory cell differentiation. We conclude from our studies in cochlear-relevant cells that the selected Cx26 mutants likely evoke hearing loss via three unique connexin defects that are independent of Cx43 status.
ABSTRACT
Characterization of the pluripotent "ground state" has led to a greater understanding of species-specific stem cell differences and has imparted an appreciation of the pluripotency continuum that exists in stem cells in vitro. Pluripotent stem cells are functionally coupled via connexins that serve in gap junctional intercellular communication (GJIC) and here we report that the level of connexin expression in pluripotent stem cells depends upon the state in which stem cells exist in vitro. Human and mouse pluripotent stem cells stabilized in a developmentally primitive or "naïve" state exhibit significantly less connexin expression compared with stem cells which are "primed" for differentiation. This dynamic connexin expression pattern may be governed, in part, by differential regulation by pluripotency transcription factors expressed in each cell state. Species-specific differences do exist, however, with mouse stem cells expressing several additional connexin transcripts not found in human pluripotent stem cells. Moreover, pharmacological inhibition of GJIC shows limited impact on naïve human stem cell survival, self-renewal, and pluripotency but plays a more significant role in primed human pluripotent stem cells. However, CRISPR-Cas9 gene ablation of Cx43 in human and mouse primed and naïve pluripotent stem cells reveals that Cx43 is dispensable in each of these four pluripotent stem cell types.
Subject(s)
Connexins/metabolism , Pluripotent Stem Cells/metabolism , Animals , Cell Communication , Cell Differentiation , Humans , MiceABSTRACT
In the last couple of decades, there has been a growing optimism surrounding the potential transformative use of human mesenchymal stem cells (MSCs) and human-induced pluripotent stem cells (iPSCs) for regenerative medicine and disease treatment. In order for this to occur, it is first essential to understand the mechanisms underpinning their cell-fate specification, which includes cell signaling via gap junctional intercellular communication. Here, we investigated the role of the prototypical gap junction protein, connexin43 (Cx43), in governing the differentiation of iPSCs into MSCs and MSC differentiation along the adipogenic lineage. We found that control iPSCs, as well as iPSCs derived from oculodentodigital dysplasia patient fibroblasts harboring a GJA1 (Cx43) gene mutation, successfully and efficiently differentiated into LipidTox and perilipin-positive cells, indicating cell differentiation along the adipogenic lineage. Furthermore, the complete CRISPR-Cas9 ablation of Cx43 from iPSCs did not prevent their differentiation into bona fide MSCs or pre-adipocytes, strongly suggesting that even though Cx43 expression is upregulated during adipogenesis, it is expendable. Interestingly, late passage Cx43-ablated MSCs senesced more quickly than control cells, resulting in failure to properly differentiate in vitro. We conclude that despite being upregulated during adipogenesis, Cx43 plays no detectable role in the early stages of human iPSC-derived MSC adipogenic differentiation. However, Cx43 may play a more impactful role in protecting MSCs from premature senescence.
Subject(s)
Adipogenesis , Cell Differentiation , Cellular Senescence , Connexin 43/metabolism , Mesenchymal Stem Cells/cytology , Connexin 43/deficiency , Gene Expression Regulation , Humans , Time FactorsABSTRACT
Hormones can transmit signals through adenosine 3',5'-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) subunits with A-kinase-anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP. Chemical-biology- and live cell-imaging techniques revealed that catalytically active PKA holoenzymes remained intact within the cytoplasm. These findings indicate that the parameters of anchored PKA holoenzyme action are much more restricted than originally anticipated.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Holoenzymes/metabolism , Signal Transduction , A Kinase Anchor Proteins/metabolism , Animals , Cell Line, Tumor , Cyclic AMP/chemistry , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/genetics , Holoenzymes/chemistry , Humans , Mice , Microscopy, Electron , Mitochondria/enzymology , Phosphorylation , Protein Binding , Protein Stability , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
We present for the first time the generation of induced pluripotent stem cells (iPSCs) from a patient with a connexin-linked disease. The importance of gap junctional intercellular communication in bone homeostasis is exemplified by the autosomal dominant developmental disorder oculodentodigital dysplasia (ODDD), which is linked to mutations in the GJA1 (Cx43) gene. ODDD is characterized by craniofacial malformations, ophthalmic deficits, enamel hypoplasia, and syndactyly. In addition to harboring a Cx43 p.V216L mutation, ODDD iPSCs exhibit reduced Cx43 mRNA and protein abundance when compared to control iPSCs and display impaired channel function. Osteogenic differentiation involved an early, and dramatic downregulation of Cx43 followed by a slight upregulation during the final stages of differentiation. Interestingly, osteoblast differentiation was delayed in ODDD iPSCs. Moreover, Cx43 subcellular localization was altered during chondrogenic differentiation of ODDD iPSCs compared to controls and this may have contributed to the more compact cartilage pellet morphology found in differentiated ODDD iPSCs. These studies highlight the importance of Cx43 expression and function during osteoblast and chondrocyte differentiation, and establish a potential mechanism for how ODDD-associated Cx43 mutations may have altered cell lineages involved in bone and cartilage development. © 2017 American Society for Bone and Mineral Research.
Subject(s)
Cell Differentiation , Connexin 43/genetics , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Mutation/genetics , Chondrogenesis , Collagen/metabolism , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/pathology , Dermis/pathology , Eye Abnormalities/genetics , Eye Abnormalities/pathology , Fibroblasts/metabolism , Foot Deformities, Congenital/genetics , Foot Deformities, Congenital/pathology , Gap Junctions/metabolism , Humans , Osteogenesis , Syndactyly/genetics , Syndactyly/pathology , Tooth Abnormalities/genetics , Tooth Abnormalities/pathologyABSTRACT
Connexins and pannexins are two families of large-pore channel forming proteins that are capable of passing small signaling molecules. While connexins serve the seminal task of direct gap junctional intercellular communication, pannexins are far less understood but function primarily as single membrane channels in autocrine and paracrine signaling. Advancements in connexin and pannexin biology in recent years has revealed that in addition to well-described classical functions at the plasma membrane, exciting new evidence suggests that connexins and pannexins participate in alternative pathways involving multiple intracellular compartments. Here we briefly highlight classical functions of connexins and pannexins but focus our attention mostly on the transformative findings that suggest that these channel-forming proteins may serve roles far beyond our current understandings.
Subject(s)
Cell Biology , Connexins/metabolism , Nerve Tissue Proteins/metabolism , Alternative Splicing/genetics , Animals , Disease , Gap Junctions/metabolism , HumansABSTRACT
Oculodentodigital dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.
Subject(s)
Connexin 43/genetics , Craniofacial Abnormalities/genetics , Eye Abnormalities/genetics , Foot Deformities, Congenital/genetics , Syndactyly/genetics , Tooth Abnormalities/genetics , Cell Communication/physiology , Cells, Cultured , Connexin 43/metabolism , Craniofacial Abnormalities/metabolism , Craniofacial Abnormalities/pathology , Eye Abnormalities/metabolism , Eye Abnormalities/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Foot Deformities, Congenital/metabolism , Foot Deformities, Congenital/pathology , Gap Junctions/metabolism , Humans , Mutation , Signal Transduction , Syndactyly/metabolism , Syndactyly/pathology , Tooth Abnormalities/metabolism , Tooth Abnormalities/pathologyABSTRACT
Pannexin1 (PANX1) is probably best understood as an ATP release channel involved in paracrine signaling. Given its ubiquitous expression, PANX1 pathogenic variants would be expected to lead to disorders involving multiple organ systems. Using whole exome sequencing, we discovered the first patient with a homozygous PANX1 variant (c.650GâA) resulting in an arginine to histidine substitution at position 217 (p.Arg217His). The 17-year-old female has intellectual disability, sensorineural hearing loss requiring bilateral cochlear implants, skeletal defects, including kyphoscoliosis, and primary ovarian failure. Her consanguineous parents are each heterozygous for this variant but are not affected by the multiorgan syndromes noted in the proband. Expression of the p.Arg217His mutant in HeLa, N2A, HEK293T, and Ad293 cells revealed normal PANX1 glycosylation and cell surface trafficking. Dye uptake, ATP release, and electrophysiological measurements revealed p.Arg217His to be a loss-of-function variant. Co-expression of the mutant with wild-type PANX1 suggested the mutant was not dominant-negative to PANX1 channel function. Collectively, we demonstrate a PANX1 missense change associated with human disease in the first report of a "PANX1-related disorder."
Subject(s)
Abnormalities, Multiple/genetics , Connexins/genetics , Germ-Line Mutation , Nerve Tissue Proteins/genetics , Abnormalities, Multiple/metabolism , Abnormalities, Multiple/pathology , Adenosine Triphosphate/metabolism , Adolescent , Animals , Cell Line, Tumor , Connexins/metabolism , Consanguinity , Family Health , Female , HEK293 Cells , HeLa Cells , Hearing Loss, Sensorineural/pathology , Heterozygote , Homozygote , Humans , Kyphosis/pathology , Male , Mutation, Missense , Nerve Tissue Proteins/metabolism , Pedigree , Primary Ovarian Insufficiency/pathology , Scoliosis/pathology , SyndromeABSTRACT
Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5ß1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43 in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells.
Subject(s)
Connexin 43/genetics , Craniofacial Abnormalities/genetics , Eye Abnormalities/genetics , Fibroblasts/metabolism , Foot Deformities, Congenital/genetics , Gene Expression Regulation , Syndactyly/genetics , Tooth Abnormalities/genetics , Animals , Cell Culture Techniques , Cells, Cultured , Connexin 43/metabolism , Craniofacial Abnormalities/metabolism , Craniofacial Abnormalities/pathology , Dermis/pathology , Disease Models, Animal , Eye Abnormalities/metabolism , Eye Abnormalities/pathology , Foot Deformities, Congenital/metabolism , Foot Deformities, Congenital/pathology , Humans , Immunoblotting , Mice , Microscopy, Confocal , Mutation , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Syndactyly/metabolism , Syndactyly/pathology , Tooth Abnormalities/metabolism , Tooth Abnormalities/pathologyABSTRACT
A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.
Subject(s)
A Kinase Anchor Proteins/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinase Type II/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinase Type I/antagonists & inhibitors , Peptides/chemistry , Protein Kinase Inhibitors/chemistry , Protein Subunits/antagonists & inhibitors , A Kinase Anchor Proteins/chemistry , A Kinase Anchor Proteins/metabolism , Amino Acid Sequence , Binding Sites/drug effects , Cell Line, Tumor , Cell Membrane Permeability , Cyclic AMP-Dependent Protein Kinase Type I/chemistry , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Cyclic AMP-Dependent Protein Kinase Type II/chemistry , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Humans , Kinetics , Molecular Sequence Data , Peptides/pharmacology , Phosphorylation , Protein Binding/drug effects , Protein Conformation , Protein Kinase Inhibitors/pharmacology , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
BACKGROUND: Alzheimer's disease (AD) pathology occurs in part as the result of excessive production of ß-amyloid (Aß). Metabotropic glutamate receptor 5 (mGluR5) is now considered a receptor for Aß and consequently contributes to pathogenic Aß signaling in AD. RESULTS: Genetic deletion of mGluR5 rescues the spatial learning deficits observed in APPswe/PS1ΔE9 AD mice. Moreover, both Aß oligomer formation and Aß plaque number are reduced in APPswe/PS1ΔE9 mice lacking mGluR5 expression. In addition to the observed increase in Aß oligomers and plaques in APPswe/PS1ΔE9 mice, we found that both mTOR phosphorylation and fragile X mental retardation protein (FMRP) expression were increased in these mice. Genetic deletion of mGluR5 reduced Aß oligomers, plaques, mTOR phosphorylation and FMRP expression in APPswe/PS1ΔE9 mice. CONCLUSIONS: Thus, we propose that Aß activation of mGluR5 appears to initiate a positive feedback loop resulting in increased Aß formation and AD pathology in APPswe/PS1ΔE9 mice via mechanism that is regulated by FMRP.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Cognition Disorders/metabolism , Cognition Disorders/pathology , Receptor, Metabotropic Glutamate 5/deficiency , Alzheimer Disease/complications , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Animals , Cell Membrane/metabolism , Cognition Disorders/complications , Cognition Disorders/physiopathology , Disease Models, Animal , Fragile X Mental Retardation Protein/metabolism , Gene Deletion , Humans , Inositol Phosphates/metabolism , Maze Learning , Memory Disorders/complications , Memory Disorders/pathology , Memory Disorders/physiopathology , Mice , Mice, Knockout , Motor Activity , Phenotype , Receptor, Metabotropic Glutamate 5/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
A-kinase anchoring proteins (AKAPs) streamline signal transduction by localizing signaling enzymes with their substrates. Great strides have been made in elucidating the role of these macromolecular signaling complexes as new binding partners and novel AKAPs are continually being uncovered. The mechanics and dynamics of these multi-enzyme assemblies suggest that AKAP complexes are viable targets for therapeutic intervention. This review will highlight recent advances in AKAP research focusing on local signaling events that are perturbed in disease.
Subject(s)
A Kinase Anchor Proteins/metabolism , A Kinase Anchor Proteins/chemistry , Animals , Humans , Molecular Targeted Therapy , Signal Transduction , Substrate SpecificityABSTRACT
Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an â¼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Chromatography, Gel , Microscopy, Electron , Phosphorylation , Substrate SpecificityABSTRACT
Rab GTPases play an essential role in the regulation of intracellular transport including the budding, tethering, and fusion of vesicles as well as organelle motility. The regulation of G protein-coupled receptor (GPCR) trafficking by Rab GTPases has traditionally been regarded as a non-specific process that facilitates the movement of the receptors between intracellular membrane compartments. Thus, alterations in GPCR signal transduction and trafficking following the overexpression of constitutively active and dominant negative Rabs were originally considered to be solely the passive by-product of perturbations in intracellular compartmental dynamics. Recently, an explosion of experimental studies has provided increasingly convincing evidence that receptor trafficking actively affects the signal transduction of cargo proteins and that the signaling of GPCR vesicular cargo can in turn modulate Rab GTPase regulated intracellular transport processes. This research is revealing how different Rabs coordinate with themselves and other regulatory molecules to mediate protein trafficking, as well as uncovers novel functions for traditional Rabs, while illustrating the active role these trafficking molecules play in pathology of disease. Recently published in the Journal of Neuroscience, Esseltine et al., present a novel role for the typified exocytic small G protein Rab8 in the intracellular trafficking and signal transduction of metabotropic glutamate receptor 1.