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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.

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.

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 (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 β-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 β-Pix and SNX27 is specific for β-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 β-Pix. Furthermore, we show recruitment of the β-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 β-Pix to focal adhesions and thereby influences cell motility.

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.

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 β (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 β or α/β 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 β, but not the α subunit. Meprin β 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 β regulates the activity of ENaC in a metalloprotease-dependent fashion.

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.

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.

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.

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.

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.

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 (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.

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.

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.

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.

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.

Although initially described as a cytosolic scaffolding protein, YAP (Yes-associated protein of 65 kDa) is known to associate with multiple transcription factors in the nucleus. Using affinity chromatography and mass spectrometry, we show that YAP interacts with heterogeneous nuclear ribonuclear protein U (hnRNP U), an RNA- and DNA-binding protein enriched in the nuclear matrix that also plays a role in the regulation of gene expression. hnRNP U interacts specifically with the proline-rich amino terminus of YAP, a region of YAP that is not found in the related protein TAZ. Although hnRNP U and YAP localize to both the nucleus and the cytoplasm, YAP does not translocate to the nucleus in an hnRNP U-dependent manner. Furthermore, hnRNP U and YAP only interact in the nucleus, suggesting that the association between the two proteins is regulated. Co-expression of hnRNP U attenuates the ability of YAP to increase the activity of a p73-driven Bax-luciferase reporter plasmid. In contrast, hnRNP U has no effect when co-expressed with a truncated YAP protein lacking the hnRNP U-binding site. Because YAP is distinguished from the homologue TAZ by its proline-rich amino terminus, the YAP-hnRNP U interaction may uniquely regulate the nuclear function(s) of YAP. The YAP-hnRNP U interaction provides another mechanism of YAP transcriptional regulation.

Spinophilin/neurabin II is an actin-associated scaffolding protein enriched in the dendritic spines of neurons. Previously, the actin-binding domain (ABD) of spinophilin was localized to a domain between amino acids (aa) 1 and 154. In a mass spectrometry screen for spinophilin-binding proteins, we have identified an additional actin-binding region between aa 151 and 282. F-actin co-sedimentation and GST affinity chromotography experiments further substantiate this result. Phalloidin staining of Rat2 fibroblasts transiently expressing GFP-spinophilin deletion constructs indicates co-localization with a subset of actin. Regions of spinophilin that lack the revised ABD (aa 1-230) do not co-localize with phalloidin-labeled actin, suggesting that the actin-binding domain contributes to directing the subcellular distribution of spinophilin. Targeting experiments using primary hippocampal cultures indicate that only the first actin-binding site contributes to dendritic spine localization. The second ABD targets to spines inefficiently and thus may interact with and affect actin filaments in a different manner than the first ABD.

The morphology and function of rat GH3 pituitary cells are profoundly affected by estradiol-17beta (E2), presumably due to changes in the profile of gene expression. We recently reported that a major target of E2 in these cells is the ezrin gene, which encodes a cytoskeletal linker protein that forms a complex with ezrin/radixin/ moesin-binding protein 50 (EBP50) in some cell types. Other studies have shown that EBP50 levels are increased by E2 in human breast and uterine tissue. Thus, we examined whether ezrin and EBP50 expression is coordinately increased by E2 in GH3 cells in vitro and rat pituitary glands in vivo. Ezrin levels are repressed by the steroidal antiestrogen, ICI 182780, and this effect is abrogated by E2 and the ERalpha-specific agonist, PPT, in GH3 cells. In contrast, EBP50 levels remained constant during these treatments. Ezrin and EBP50 did not display extensive colocalization. Moreover, ezrin was not co-immunoprecipitated by an EBP50 antibody in parental GH3 cells or in GH3 cells stably overexpressing EBP50, but was co-immunoprecipitated with EBP50 in human breast MCF-7 cells. Disruption of the actin cytoskeleton of GH3 cells changed the distribution of ezrin within subcellular fractions, but had no effect on EBP50. Finally, in juvenile female rats, E2 injections increased ezrin expression in the pituitary and uterus, but increased EBP50 expression only in the uterus. These findings demonstrate tissue specificity in the formation of ezrin-EBP50 complexes and in the regulation of EBP50 expression in estrogen-responsive tissues.

Defects in the trafficking of apical membrane proteins in polarized epithelial cells are often associated with diseases, including cystic fibrosis, Liddle's syndrome, nephrogenic diabetes insipidus and Dubin-Johnson syndrome. In recent years, we have learned much about the specialized apical trafficking pathways in polarized cells. Many laboratories have identified signals that direct proteins within these pathways and have defined protein interactions that mediate specific steps in the sorting and stabilization of these proteins. In addition, many cytosolic proteins, including lipid kinases, GTPases, ATPases and scaffolding/adaptor proteins that lack enzymatic activity, regulate the trafficking of proteins through these pathways. Recent advances in the field include the role of small GTPases, unconventional myosins and lipid kinases in apical endocytosis and transcytosis, and the identification of PDZ proteins that regulate apical membrane trafficking of receptors, transporters and ion channels.

MRP2, a member of the ABC protein superfamily, functions as an ATP-dependent export pump for anionic conjugates in the apical membranes of epithelial cells. It has been reported that the trafficking of MRP2 is modulated by PKC. Adjacent to the C-terminal PDZ binding motif, which may be involved in the targeting of MRP2, we found a potential PKC phosphorylation site (Ser(1542)). Therefore, we examined the interaction of MRP2 and its phosphorylation-mimicking mutants with different PDZ proteins (EBP50, E3KARP, PDZK1, IKEPP, beta2-syntrophin, and SAP-97). The binding of these PDZ proteins to CFTR and ABCA1, other ABC proteins, possessing PDZ binding motif, was also studied. We observed a strong binding of apically localized PDZ proteins to both MRP2 and CFTR, whereas beta2-syntrophin exhibited binding only to ABCA1. The phosphorylation-mimicking MRP2 mutant and a phosphorylated C-terminal MRP2 peptide showed significantly increased binding to IKEPP, EBP50, and both individual PDZ domains of EBP50. Our results suggest that phosphorylation of the MRP2 PDZ binding motif has a profound effect on the PDZ binding of MRP2.

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents., (Copyright 2003 John Wiley & Sons, Ltd.)

Although protein kinase A (PKA) activation is known to increase ciliary beat frequency in humans the molecular mechanisms involved are unknown. We demonstrate that PKA is associated with ciliary axonemes where it specifically phosphorylates a 23-kDa protein. Because PKA is often localized to subcellular compartments in proximity to its substrate(s) via interactions with A-kinase-anchoring proteins (AKAPs), we investigated whether an AKAP was also associated with ciliary axonemes. This study has identified a novel 28 kDa AKAP (AKAP28)that is highly enriched in airway axonemes. The mRNA for AKAP28 is up-regulated as primary airway cells differentiate and is specifically expressed in tissues containing cilia and/or flagella. Additionally, both Western blot and immunostaining data show that AKAP28 is enriched in airway cilia. These data demonstrate that we have identified the first human axonemal AKAP, a protein that likely plays a role in the signaling necessary for efficient modulation of ciliary beat frequency.

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1gamma1 and PP1alpha but not PP1beta. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1gamma1 and PP1alpha from brain extracts and associated poorly with PP1beta. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1beta and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1gamma1 > PP1alpha > PP1beta. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1gamma null mice. In the absence of PP1gamma1, both neurabins showed enhanced association with PP1alpha but not PP1beta. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1gamma1 and PP1alpha to the mammalian synapse.

Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin.

The dramatic increase in genomic information is allowing the rapid identification of genes that are altered in mental retardation (MR). It is necessary to place their resulting gene products in their cellular context to understand how they may have contributed to a patient's cognitive deficits. This review will consider signaling molecules that have been implicated in X-linked MR and the known pathways by which these proteins covey information will be delineated. The proteins discussed include four distinct classes: transmembrane receptors, guanine nucleotide related proteins, kinases, and translational regulators.

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.

Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl(-) conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A(2B) adenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to "flush" noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.

The epithelial Na(+) channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (alpha, beta, and gamma) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET(B) receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition.

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Increasing evidence suggests that protein-protein interaction is essential in many biological processes including epithelial transport. In this report, we discuss the significance of protein interactions to HCO(3)(-) secretion in pancreatic duct cells. In pancreatic ducts HCO(3)(-) secretion is mediated by cystic fibrosis transmembrane conductance regulator (CFTR) activated luminal Cl(-)/HCO(3)(-) exchange activity and HCO(3)(-) absorption is achieved by Na(+)-dependent mechanisms including Na(+)/H(+) exchanger 3 (NHE3). We found biochemical and functional association between CFTR and NHE3. In addition, protein binding through PDZ modules is needed for this regulatory interaction. CFTR affected NHE3 activities in two ways. Acutely, CFTR augmented the cAMP-dependent inhibition of NHE3. In a chronic mechanism, CFTR increases the luminal expression of Na(+)/H(+) exchange in pancreatic duct cells. These findings reveal that protein complexes in the plasma membrane of pancreatic duct cells are highly organized for efficient HCO(3)(-) secretion.

The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO3- secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO3- secretion, the role of CFTR in HCO3- salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO3- salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote DeltaF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of DeltaF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of DeltaF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO3- homeostasis by regulating both pancreatic ductal HCO3- secretory and salvage mechanisms.

Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 +/- 0.12 to 1.23 +/- 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.

We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na(+)/H(+) exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50-YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.

Signaling through D2 class dopamine receptors is crucial to correct brain development and function, and dysfunction of this system is implicated in major neurological disorders such as Parkinson's disease and schizophrenia. To investigate potential novel mechanisms of D2 receptor regulation, the third cytoplasmic loop of the D2 dopamine receptor was used to screen a rat hippocampal yeast two-hybrid library. Spinophilin, a recently characterized F-actin and protein phosphatase-1-binding protein with a single PDZ domain was identified as a protein that specifically associates with this region of D2 receptors. A direct interaction between spinophilin and the D2 receptor was confirmed in vitro using recombinant fusion proteins. The portion of spinophilin responsible for interacting with the D2 third cytoplasmic loop was narrowed to a region that does not include the actin-binding domain, the PDZ domain, or the coiled-coil. This region is distinct from the site of interaction with protein phosphatase-1, and both D2 receptors and protein phosphatase-1 may bind spinophilin at the same time. The interaction is not mediated via the unique 29-amino acid insert in D2long; both D2long and D2short third cytoplasmic loops interact with spinophilin in vitro and in yeast two-hybrid assays. Expression of D2 receptors containing an extracellular hemagglutinin epitope in Madin-Darby canine kidney cells results in co-localization of receptor and endogenous spinophilin as determined by immunocytochemistry using antibodies directed against spinophilin and the HA tag. We hypothesize that spinophilin is important for establishing a signaling complex for dopaminergic neurotransmission through D2 receptors by linking receptors to downstream signaling molecules and the actin cytoskeleton.

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.

Protein kinase A-anchoring proteins (AKAPs) localize the second messenger response to particular subcellular domains by sequestration of the type II protein kinase A. Previously, AKAP120 was identified from a rabbit gastric parietal cell cDNA library; however, a monoclonal antibody raised against AKAP120 labeled a 350-kDa band in Western blots of parietal cell cytosol. Recloning has now revealed that AKAP120 is a segment of a larger protein, AKAP350. We have now obtained a complete sequence of human gastric AKAP350 as well as partial cDNA sequences from human lung and rabbit parietal cells. The genomic region containing AKAP350 is found on chromosome 7q21 and is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, a protein associated with the N-methyl-D-aspartate receptor. Rabbit parietal cell AKAP350 is missing a sequence corresponding to a single exon in the middle of the molecule located just after the yotiao homology region. Two carboxyl-terminal splice variants were also identified. Both of the major splice variants showed tissue- and cell-specific expression patterns. Immunofluorescence microscopy demonstrated that AKAP350 was associated with centrosomes in many cell types. In polarized Madin-Darby canine kidney cells, AKAP350 localized asymmetrically to one pole of the centrosome, and nocodazole did not alter its localization. During the cell cycle, AKAP350 was associated with the centrosomes as well as with the cleavage furrow during anaphase and telophase. Several epithelial cell types also demonstrated noncentrosomal pools of AKAP350, especially parietal cells, which contained multiple cytosolic immunoreactive foci throughout the cells. The localization of AKAP350 suggests that it may regulate centrosomal and noncentrosomal cytoskeletal systems in many different cell types.

The cytosolic domain of the peptide processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) contains signals that direct its trafficking in the secretory and endosomal pathways. Using the yeast two-hybrid system, Alam et al. (Alam, M. R., Caldwell, B. D., Johnson, R. C., Darlington, D. N., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 28636) identified three proteins that interact with a fragment of the PAM cytosolic domain containing these targeting signals. We cloned the rat and human cDNAs encoding PAM COOH-terminal interactor protein-1 (P-CIP1). Both cDNAs contain an open reading frame that encodes a novel protein of 435 amino acids. The P-CIP1 protein is highly conserved from rat to human (85% identity) but does not display significant homology to proteins in the GenBank data base. In vitro, P-CIP1 interacts with the cytosolic domain of wild type PAM-1, but does not interact with mutant PAM-1 proteins that fail to target correctly when expressed in endocrine cells. P-CIP1 contains multiple consensus serine/threonine phosphorylation sites and a region predicted to form a coiled-coil at the COOH terminus. When expressed in endocrine cells or fibroblasts, P-CIP1 is distributed in a punctate pattern in the perinuclear area but does not significantly overlap the distribution of transfected wild type PAM-1. The distribution of P-CIP1 displays significant overlap with the distribution of the secretory carrier membrane proteins, internalized Texas Red-conjugated transferrin, and Rab11. The data suggest that P-CIP1 associates with vesicles in the recycling endosomal pathway, and may play a role in regulating the trafficking of integral membrane PAM.

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl- channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl- channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl- channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins.

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.

We have investigated the trafficking of integral membrane peptidylglycine alpha-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 cell line. This bifunctional enzyme has two domains which together catalyze the COOH-terminal alpha-amidation of peptidylglycine substrates yielding amidated products stored in secretory granules. As soluble proteins, both catalytic domains were independently targeted to secretory granules. In contrast, membrane PAM was largely localized to the trans-Golgi network (TGN). Upon truncation of its cytoplasmic COOH-terminal domain, membrane PAM was less efficiently cleaved by secretory granule enzymes and accumulated on the plasma membrane. When transferred to the lumenal domain of the interleukin 2 receptor alpha-chain (Tac protein), the cytoplasmic domain of PAM caused rerouting of Tac from the surface to the TGN and supported internalization of Tac antibody from the plasma membrane. To define sequences in the cytoplasmic domain of integral membrane PAM involved in its trafficking, we expressed PAM proteins containing truncations, deletions, or point mutations in the COOH-terminal cytoplasmic domain. PAM proteins were not retained in the TGN when half of the cytoplasmic domain was deleted; such proteins accumulated on the plasma membrane, were not efficiently internalized, and were cleaved to generate a bifunctional PAM protein that was not stored in secretory granules. A tyrosine-based internalization motif was identified, which was not required for efficient cleavage of full-length integral membrane PAM by secretory granule enzymes. Deletion of an 18-amino acid domain surrounding this Tyr residue both diminished cleavage of membrane PAM by secretory granule enzymes and eliminated internalization of PAM from the plasma membrane. The cytoplasmic domain is responsible for retaining membrane PAM in the TGN and for retrieving membrane PAM from the cell surface, while the lumenal catalytic domains of PAM appear to be responsible for targeting the protein to secretory granules.

To investigate factors governing proteolytic processing and routing of biologically active peptides in the secretory pathway, cDNAs for preproneuropeptide Y (preproNPY) and preproneuropeptide Y fused to a membrane anchor were transfected into pituitary cells. The anchor was the transmembrane and COOH-terminal cytoplasmic domain of peptidylglycine alpha-amidating monooxygenase (PAM); these domains are essential for correct routing of integral membrane forms of PAM. Like proneuropeptide Y (proNPY), the integral membrane form of proNPY was a good substrate for the endogenous prohormone convertases, yielding soluble NPY stored in regulated secretory granules. Tethering of proNPY to the membrane resulted in only a small delay in the rate of cleavage to produce mature NPY and in the arrival of NPY in regulated secretory granules. In contrast, the COOH-terminal region of proNPY remained attached to the transmembrane/COOH-terminal domain of PAM and was rerouted to the vicinity of the trans-Golgi network, where integral membrane forms of PAM are concentrated. Thus, the COOH-terminal of proNPY cannot override the signals in the PAM membrane anchor.

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within cells including endocrine cells. Several isoforms (I, II, and III) of IP3Rs have been identified, which are encoded by separate genes, and are expressed in many tissues with differing patterns of cellular expression. We have generated specific affinity-purified polyclonal anti-peptide antibodies to each of the three isoforms. Western blot analysis of RINm5F and ATt20 cells shows high levels of endogenously expressed type I and type III IP3R, but undetectable levels of type II. Immunofluorescence studies revealed an endoplasmic reticulum-like pattern similar to BiP, an ER marker. In contrast with previous claims, both type I and type III IP3Rs were absent from the secretory granules of ATt20 cells. Western blots of sucrose gradients and gel filtration probed with antibodies to either type I or type III showed a molecular weight of greater than 1,000 kDa consistent with a tetrameric structure. Co-immunoprecipitation experiments indicated that most of the receptors were present as heterotetramers. Homotetramers were identified for the type III IP3R; however, type I homotetramers were undetectable. These data suggest that molecular association of IP3Rs into heterotetrameric forms can contribute to the complexity of the regulation of Ca2+ release from ER by IP3Rs within cells.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the COOH-terminal alpha-amidation of neural and endocrine peptides through a two-step reaction carried out sequentially by its monooxygenase and lyase domains. PAM occurs in soluble and integral membrane forms. Metabolic labeling of stably transfected hEK-293 and AtT-20 cells showed that [32P]PO4(3-) was efficiently incorporated into Ser and Thr residues of membrane PAM but not into soluble PAM. Truncation of integral membrane PAM proteins (which terminate with Ser976) at Tyr936 eliminated their phosphorylation, suggesting that the COOH-terminal region of the protein was the site of phosphorylation. Recombinant PAM COOH-terminal domain was phosphorylated on Ser932 and Ser937 by protein kinase C (PKC). PAM-1 protein recovered from different subcellular fractions of stably transfected AtT-20 cells was differentially susceptible to calcium-dependent, staurosporine-inhibitable phosphorylation catalyzed by endogenous cytosolic protein kinase(s). Although phorbol ester treatment of hEK-293 cells expressing PAM-1 stimulated the cleavage/release of a bifunctional 105-kDa PAM protein, the effect was an indirect one since it was also observed in hEK-293 cells expressing a truncated PAM-1 protein that was not phosphorylated. AtT-20 cells expressing PAM-1 lacking one of the PKC sites (PAM-1/Ser937-->Ala) exhibited an altered pattern of PAM.PAM antibody internalization, with the mutant protein targeted to lysosomes upon internalization. Thus, phosphorylation of Ser937 in the COOH-terminal cytosolic domain of membrane PAM plays a role in a specific step in the targeting of this protein.