Becoming a resilient scientist series: An intervention program.

Compared to the general population, science trainees experience challenges and heightened stressors that often lead to adverse mental health outcomes. With COVID-19, the stressors of social distancing, isolation, truncated lab time, and uncertainty about the future have all likely exacerbated these issues. Now, more than ever, practical and effective interventions are vitally needed to address the core causes of stress among science trainees and increase their resilience. This paper introduces a new resilience program targeted to biomedical trainees and scientists - Becoming a Resilient Scientist Series (BRS), a multi-part workshop complemented by facilitated group discussions all aimed at bolstering resilience, particularly in the context of academic and research environments. To assess the program's efficacy, participants completed resilience measures and related assessments before and after completing the series. The results demonstrate that BRS significantly enhances trainee resilience (primary outcome) and reduces perceived stress, anxiety, and work-related presenteeism, as well as increased adaptability, self-awareness, and self-efficacy (secondary outcomes). Furthermore, program participants reported a high level of satisfaction, a strong willingness to recommend the program to others, and perceived positive changes in their resilience skills. To the best of our knowledge, this is the first resilience program designed explicitly for biomedical trainees and scientists, tailored to their unique professional culture and work environment.

Sorting nexin 27 (SNX27) associates with zonula occludens-2 (ZO-2) and modulates the epithelial tight junction.

Proteins of the SNX (sorting nexin) superfamily are characterized by the presence of a PX (Phox homology) domain and associate with PtdIns3P (phosphatidylinositol-3-monophosphate)-rich regions of the endosomal system. SNX27 is the only sorting nexin that contains a PDZ domain. In the present study, we used a proteomic approach to identify a novel interaction between SNX27 and ZO-2 [zonula occludens-2; also known as TJP2 (tight junction protein 2)], a component of the epithelial tight junction. The SNX27-ZO-2 interaction requires the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of ZO-2. When tight junctions were perturbed by chelation of extracellular Ca2+, ZO-2 transiently localized to SNX27-positive early endosomes. Depletion of SNX27 in mpkCCD (mouse primary kidney cortical collecting duct) cell monolayers resulted in a decrease in the rate of ZO-2, but not ZO-1, mobility at cell-cell contact regions after photobleaching and an increase in junctional permeability to large solutes. The findings of the present study identify an important new SNX27-binding partner and suggest a role for endocytic pathways in the intracellular trafficking of ZO-2 and possibly other tight junction proteins. Our results also indicate a role for SNX27-ZO-2 interactions in tight junction maintenance and function.

Dileucine and PDZ-binding motifs mediate synaptic adhesion-like molecule 1 (SALM1) trafficking in hippocampal neurons.

Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.

Sorting nexin 27 protein regulates trafficking of a p21-activated kinase (PAK) interacting exchange factor (ß-Pix)-G protein-coupled receptor kinase interacting protein (GIT) complex via a PDZ domain interaction.

Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that ß-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of ß-Pix and SNX27 is specific for ß-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by ß-Pix. Furthermore, we show recruitment of the ß-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of ß-Pix to focal adhesions and thereby influences cell motility.

Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone.

Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.

Activation of the epithelial sodium channel by the metalloprotease meprin ß subunit.

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin ß (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin ß or α/ß in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin ß, but not the α subunit. Meprin ß promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin ß regulates the activity of ENaC in a metalloprotease-dependent fashion.

Cystic fibrosis transmembrane conductance regulator interacts with multiple immunoglobulin domains of filamin A.

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa.partner complexes as templates, we generated in silico models of IgFLNa.CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated ( approximately 160 nm) strands, whereas CFTR is compact (6 approximately 8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa.partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.

Isoform divergence of the filamin family of proteins.

The vertebrate filamin family (A, B, and C) is part of the spectrin family of actin cross-linking proteins. Family members share high sequence similarity (>64%) and have both common and isoform-distinct functionalities. To identify the basis for isoform-specific functionality, we perform an evolutionary trace of chordate filamin at the granularity of single residues. Our trace methodology is constrained to focus on neofunctionality by requiring that one isoform remain the ancestral type, whereas at least one isoform has an accepted mutation. We call divergence meeting these characteristics "class-distinctive." To obtain a temporal and spatial context for class-distinctive residues, we derive an all-atom model of full-length filamin A by homology modeling and joining individual domains. We map onto our model both conserved and class-distinctive residues along with the period (Teleostei, Amphibian, and Mammalian) in which they diverged. Our phylogenetic analysis suggests that filamins diverged from a common ancestral gene between urochordate and vertebrate lineages. Filamins also diverged the most just after gene duplication, in the Teleostei period, with filamin C remaining closest to ancestral filamin. At the residue level, domains with well-characterized interfaces, IgFLN 17 and IgFLN 21 (immunoglobulin, Ig), have diverged in potentially critical residues in their adhesion protein-binding interfaces, signifying that isoforms may bind or regulate ligand binding differentially. Similarly, isoform divergence in a region associated with F actin-binding regulation suggests that isoforms differentially regulate F-actin binding. In addition, we observe some class-distinctive residues in the vicinity of missense mutations that cause filamin A and B-associated skeletal disorders. Our analysis, utilizing both spatial and temporal granularity, has identified potentially important residues responsible for vertebrate filamin isoform-specific divergence-significantly in regions where few binding partners have been discovered to date- and suggests yet to be discovered filamin-binding partners and isoform-specific differential regulation with these binding partners.

Elevated intracellular calcium stimulates NHE3 activity by an IKEPP (NHERF4) dependent mechanism.

The ileal brush border (BB) contains four evolutionarily related multi-PDZ domain proteins including NHERF1, NHERF2, PDZK1 (NHERF3) and IKEPP (NHERF4). Why multiple related PDZ proteins are in a similar location in the same cell is unknown. However, some specificity in regulation of NHE3 activity has been identified. For example, elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2 but not NHERF1, and involves the formation of large NHE3 complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether the four PDZ domain containing protein IKEPP reconstitutes elevated [Ca(2+)](i) regulation of NHE3. In vitro, IKEPP bound to the F2 region (aa 590-667) of NHE3 in overlay assays, which is the same region where NHERF1 and NHERF2 bind. PS120 cells lack endogenous NHE3 and IKEPP. Treatment of PS120/NHE3/IKEPP cells (stably transfected with NHE3 and IKEPP) with the Ca(2+) ionophore, 4-Br-A23187 (0.5 microM), stimulated NHE3 V(max) activity by approximately 40%. This was associated with an increase in plasma membrane expression of NHE3 by a similar amount. NHE3 activity and surface expression were unaffected by A23187 in PS120/NHE3 cells lacking IKEPP. Based on sucrose density gradient centrifugation, IKEPP was also shown to exist in large complexes, some of which overlap in size with NHE3, and the size of both NHE3 and IKEPP complexes decreased in parallel after [Ca(2+)](i) elevation. FRET experiments on fixed cells demonstrated that IKEPP and NHE3 directly associated at an intracellular site. Elevating [Ca(2+)](i) decreased this intracellular NHE3 and IKEPP association. In summary: (1) In the presence of IKEPP, elevated [Ca(2+)](i) stimulates NHE3 activity. This was associated with increased expression of NHE3 in the plasma membrane as well as a shift to smaller sizes of NHE3 and IKEPP containing complexes. (2) IKEPP directly binds NHE3 at its F2 C-terminal domain and directly associates with NHE3 in vivo (FRET). (3) Elevated [Ca(2+)](i) decreased the association of IKEPP and NHE3 in an intracellular compartment. Based on which NHERF family member is expressed in PS120 cells, elevated [Ca(2+)](i) stimulates (IKEPP), inhibits (NHERF2) or does not affect (NHERF1) NHE3 activity. This demonstrates that regulation of NHE3 depends on the nature of the NHERF family member associating with NHE3 and the accompanying NHE3 complexes.

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation.

Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides.

How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol-anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle-induced clusters of both Thy-1 and CD73, a 5' exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.

Polarized apical sorting of guanylyl cyclase C is specified by a cytosolic signal.

Receptor guanylyl cyclases respond to ligand stimulation by increasing intracellular cGMP, thereby initiating a variety of cell-signaling pathways. Furthermore, these proteins are differentially localized at the apical and basolateral membranes of epithelial cells. We have identified a region of 11 amino acids in the cytosolic COOH terminus of guanylyl cyclase C (GCC) required for normal apical localization in Madin-Darby canine kidney (MDCK) cells. These amino acids share no significant sequence homology with previously identified cytosolic apical sorting determinants. However, these amino acids are highly conserved and are sufficient to confer apical polarity to the interleukin-2 receptor alpha-chain (Tac). Additionally, we find two molecular weight species of GCC in lysates prepared from MDCK cells over-expressing GCC but observe only the fully mature species on the cell surface. Using pulse-chase analysis in polarized MDCK cells, we followed the generation of this mature species over time finding it to be detectable only at the apical cell surface. These data support the hypothesis that selective apical sorting can be determined using short, cytosolic amino acid motifs and argue for the existence of apical sorting machinery comparable with the machinery identified for basolateral protein traffic.

Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65.

YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yap(tm1Smil) allele (Yap-/-) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostaining, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in Yap-/- embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper. Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap-/- embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithelium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation.

The cystic fibrosis transmembrane conductance regulator is regulated by a direct interaction with the protein phosphatase 2A.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aalpha and B'epsilon and find that the B'epsilon subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies.

Beyond the brush border: NHERF4 blazes new NHERF turf.

The Na exchanger regulatory factor (NHERF) family of epithelial-enriched PDZ domain scaffolding proteins plays important roles in maintaining and regulating epithelial cell function. The NHERFs exhibit some overlap in tissue distribution and binding partners, suggesting redundant functions. Yet, it is clear that each NHERF protein exhibits distinct properties, translating into unique cellular functions. The work summarized in this review suggests the most recently identified family member, NHERF4, is the most divergent. Additional investigation is needed, however, to understand more completely the role of NHERF4 in the context of the NHERF family.

Improved protein identification through the use of unstained gels.

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.

Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors.

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Galpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellular loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of alpha-adrenergic receptor (alphaAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alphaAR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of betaAR-alphaAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alphaAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2-/- cells and less sensitive to inhibition by RGS2 in spl-/- cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.

Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium.

We demonstrated previously that Calu-3 airway epithelial cells sense adenosine on their luminal surface through adenosine A2B receptors coupled to adenylyl cyclase. Occupancy of these receptors leads to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel through protein kinase A (PKA) anchored at the apical membrane. Because luminal A2B receptor activation does not raise total cellular cAMP levels, we hypothesized that activation of phosphodiesterases (PDEs) confines cAMP generated by apical A2B receptors to a microdomain that includes the CFTR channel. Using reverse transcription-PCR, Western blotting, and activity measurements, PDE4D was identified as the major PDE species in airway epithelia. Consistent with these results, inhibitors of PDE4, but not PDE3, selectively abolished the lateral confinement of cAMP signaling in apical membrane patches during cell-attached recordings. Furthermore, stimulation of the CFTR in excised apical patches by rolipram and RS25344 indicated that PDE4 is localized in close proximity to the CFTR channel. Indeed, immunohistochemistry of human airway sections revealed that PDE4D is localized in the apical domain of the cell. PDE4 was activated after luminal adenosine exposure in a PKA-dependent manner. Because PDE4 activity is positively regulated by PKA, our results support a model whereby the PDE diffusion barrier is proportional to the degree of receptor stimulation. These findings underscore the concept that subcellular localization of individual PDE isozymes is an important mechanism for confining cAMP signaling to functional domains within cells.

The GABA transporter GAT1 and the MAGUK protein Pals1: interaction, uptake modulation, and coexpression in the brain.

GABAergic signaling in the CNS is terminated in part through uptake of GABA by GABA transporters. We used the yeast two-hybrid system to identify proteins that associate with the carboxy-terminus of the neuronal GABA transporter GAT1. We found an interaction between GAT1 and the MAGUK protein Pals1. When coexpressed in COS-7 cells, Pals1 co-immunoprecipitates with GAT1. We demonstrate cellular coexpression of GAT1 and Pals1 in the mouse hippocampus and striatum. Functionally, coexpression of GAT1 and Pals1 in COS-7 cells increases [3H]-GABA uptake by GAT1. The mechanism underlying increased uptake is increased levels of GAT1 protein. We hypothesize that Pals1 contributes to the stability of the GAT1, thus promoting the expression level of the transporter protein. In the CNS, Pals1 may stabilize GAT1 at appropriate levels in specific GABAergic neurons.