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.

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.Cystic fibrosis (CF) 1 is a lethal autosomal recessive disease characterized by defects in epithelial ion transport (1). CF is caused by mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a cAMP-regulated Cl Ϫ channel at the apical cell surface (1-3). The CF phenotype includes changes in cellular processes distinct from those involving Cl Ϫ transport, including sodium hyperabsorption and abnormalities in the processing of mucins (4 -6). The most common cause of CF are mutations that lead to the formation of an abnormally folded CFTR protein that does not reach the cell surface (2). Even wild type CFTR is inefficiently transported to the cell surface, with up to 70% of the newly synthesized proteins failing to achieve a stable conformation that escapes quality control mechanisms in the endoplasmic reticulum (2,7,8). Knowledge of the protein-protein interactions that are involved in CFTR-mediated regulation of other epithelial transport proteins, and the interactions that control the trafficking, localization, and regulation of CFTR, is incomplete. Recently, the amino terminus of CFTR was shown to interact with syntaxin 1, with implications both for insertion of CFTR into the plasma membrane and regulation of channel activity (9). Other interactions that stabilize CFTR or regulate its function remain to be identified.Compartmentalization of CFTR in a multiprotein complex might regulate CFTR activity by stabilizing the protein at the cell surface or by increasing the efficiency by which kinases and phosphatases control the channel. The presence of such a complex may also explain how CFTR modulates the activity of other epithelial cell transport proteins. A common mechanism to establish multiprotein complexes is via protein-protein interactions w...

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.

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 A2B 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.A irways continuously remove noxious materials through a mucociliary clearance process that requires liquid secretion (1, 2). cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl Ϫ channels (3, 4) are expected to participate in liquid secretion in airways, but the two key events in the activation of CFTR by local physical stimuli remain puzzling. First, how do physical stimuli initiate the classic cAMP signaling cascade, a process that is tightly regulated by Gprotein-coupled receptors (5)? Second, how does cAMP reach CFTR in the apical membrane? The dogma of G-proteincoupled receptors and adenylyl cyclase (AC) restricted to the basolateral membrane of epithelia does not adequately explain how these events occur (6).Two ongoing areas of research suggest a potentially more relevant, but as yet not fully tested model for activation of apical CFTR by a local physical stimulus. Airway surface epithelia are poorly innervated, suggesting that mucociliary clearance is subject to autocrine͞paracrine control. A leading candidate for mediating mucociliary clearance is the release of cellular nucleotides, because release occurs in response to physical stimuli and luminal nucleotide receptors stimulate apical Cl Ϫ conductance, mucus secretion, and ciliary beating (7,8). The action of ectonucleotidases extends the signaling potential of released ATP on luminal surface by producing adenosine (Ado), a ligand for A2 receptors that couple to AC (9, 10). Recent reports indicate that receptors, intracellular signaling pathways, and scaffolding molecules can form complexes that locally regulate functions in subcellular compartments (11,12). Thus, a model linking a luminal physical stimulus to activation of CFTR requires specific elements, including ATP release, ectonucleotidases, Ado receptors, G proteins, and AC, to be intimately associated with the apical cell membrane. The goal of the present study was to test this hypothesis in polarized airway epithelial cells. MethodsCells. Human Calu-3 cells were grown as previously described (13) on Costar clear transwells (for HPLC and cAMP assay) or homemade permeable ...

Abstract. Peptidylglycine o~-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and midregion lyase act in sequence to perform the peptide a-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry.Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membraneassociated were all packaged into regulated secretory granules in AtT-20 cells.

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.

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.Transduction of signals from extracellular stimuli is most commonly accomplished via ligand-receptor binding and generation of a second messenger response. While increases in intracellular second messengers have traditionally been viewed as global cellular events, second messenger effects are often limited to particular regions or organelles within cells. Investigations over the past decade have led to a greater understanding of the mechanisms responsible for the compartmentalization of second messenger effects. These studies have identified a diverse group of scaffolding proteins that sequester both protein kinases and protein phosphatases within specific cellular domains (1, 2). In the case of cAMP-dependent protein kinases, protein kinase A-anchoring proteins (AKAPs) 1 tether the protein kinase A holoenzyme through binding to the regulatory subunit dimer. A growing group of AKAPs that bind the regulatory subunit of type II protein kinase A (R II ) have been reported over the past several years. The first R II -binding protein was identified over 15 years ago when microtubule-associated protein 2 (MAP-2) was described (3, 4). Since that time, several AKAPs have been identified, localizing the type...

We have investigated the trafficking of integral membrane peptidylglycine ␣-amidating monooxygenase (PAM) in the neuroendocrine AtT-20 cell line. This bifunctional enzyme has two domains which together catalyze the COOH-terminal ␣-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 ␣-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.