Involvement of SAP97 anchored multiprotein complexes in regulating cardiorenal signaling and trafficking networks.

SAP97 is a member of the MAGUK family of proteins, but unlike other MAGUK proteins that are selectively expressed in the CNS, SAP97 is also expressed in peripheral organs, like the heart and kidneys. SAP97 has several protein binding cassettes, and this review will describe their involvement in creating SAP97-anchored multiprotein networks. SAP97-anchored networks localized at the inner leaflet of the cell membrane play a major role in trafficking and targeting of membrane G protein-coupled receptors (GPCR), channels, and structural proteins.

Involvement of PDZ-SAP97 interactions in regulating AQP2 translocation in response to vasopressin in LLC-PK cells.

Arginine-vasopressin (AVP)-mediated translocation of aquaporin-2 (AQP2) protein-forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for the renal conservation of water. The type-1 PDZ-binding motif (PBM) in AQP2, "GTKA," is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remain obscure. We determined that synapse-associated protein-97 (SAP97), a membrane-associated guanylate kinase protein involved in establishing epithelial cell polarity, was an avid binding partner to the PBM of AQP2.

Novel Paradigms Governing -Adrenergic Receptor Trafficking in Primary Adult Rat Cardiac Myocytes.

The -adrenergic receptor (-AR) is a major cardiac G protein-coupled receptor, which mediates cardiac actions of catecholamines and is involved in genesis and treatment of numerous cardiovascular disorders. In mammalian cells, catecholamines induce the internalization of the -AR into endosomes and their removal promotes the recycling of the endosomal -AR back to the plasma membrane; however, whether these redistributive processes occur in terminally differentiated cells is unknown. Compartmentalization of the -AR in response to -agonists and antagonists was determined by confocal microscopy in primary adult rat ventricular myocytes (ARVMs), which are terminally differentiated myocytes with unique structures such as transverse tubules (T-tubules) and contractile sarcomeres.

Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy.

G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation.

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes.

Two barcodes encoded by the type-1 PDZ and by phospho-Ser regulate retromer/WASH-mediated sorting of the ß-adrenergic receptor from endosomes to the plasma membrane.

Recycling of the majority of agonist-internalized GPCR is dependent on a type I-PDZ "barcode" in their C-tail. The recycling of wild-type (WT) ß-AR is also dependent on its default "type-1 PDZ barcode", but trafficking of the ß-AR is inhibited when PKA or its substrate serine at position 312 (Ser) are inactivated. We tested the hypothesis that phospho-Ser provided a second barcode for ß-AR sorting from endosomes to the plasma membrane by determining the role of retromer/WASH complexes in ß-AR trafficking.

Identification of novel transplantable GPCR recycling motif for drug discovery.

β-Adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The β-AR like other G protein-coupled receptors (GPCRs) are endocytosed in response to intense agonist activation. Recycling of the agonist-internalized β-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine in the third intracellular loop of the β-AR.

Nedd4 haploinsufficient mice display moderate insulin resistance, enhanced lipolysis, and protection against high-fat diet-induced obesity.

Neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) is the prototypical protein in the Nedd4 ubiquitin ligase (E3) family, which governs ubiquitin-dependent endocytosis and/or degradation of plasma membrane proteins. Loss of Nedd4 results in embryonic or neonatal lethality in mice and reduced insulin/IGF-1 signaling in embryonic fibroblasts. To delineate the roles of Nedd4 in vivo, we examined the phenotypes of heterozygous knockout mice using a high-fat diet-induced obesity (HFDIO) model.

Carvedilol reverses cardiac insufficiency in AKAP5 knockout mice by normalizing the activities of calcineurin and CaMKII.

Aims: Cardiac β-adrenergic receptors (β-AR) are key regulators of cardiac haemodynamics and size. The scaffolding protein A-kinase anchoring protein 79/150 (AKAP5) is a key regulator of myocardial signalling by β-ARs. We examined the function of AKAP5 in regulating cardiac haemodynamics and size, and the role of β-ARs and Ca(2+)-regulated intracellular signalling pathways in this phenomenon.

Sorting of β1-adrenergic receptors is mediated by pathways that are either dependent on or independent of type I PDZ, protein kinase A (PKA), and SAP97.

The β1-adrenergic receptor (β1-AR) is a target for treatment of major cardiovascular diseases, such as heart failure and hypertension. Recycling of agonist-internalized β1-AR is dependent on type I PSD-95/DLG/ZO1 (PDZ) in the C-tail of the β1-AR and on protein kinase A (PKA) activity (Gardner, L. A.

Role of AKAP79/150 protein in β1-adrenergic receptor trafficking and signaling in mammalian cells.

Protein kinase A-anchoring proteins (AKAPs) participate in the formation of macromolecular signaling complexes that include protein kinases, ion channels, effector enzymes, and G-protein-coupled receptors. We examined the role of AKAP79/150 (AKAP5) in trafficking and signaling of the β1-adrenergic receptor (β1-AR). shRNA-mediated down-regulation of AKAP5 in HEK-293 cells inhibited the recycling of the β1-AR.

Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs.

Background: Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin.

Methods: Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16-20 weeks of exercise training.

Adult epicardial fat exhibits beige features.

Context: Human epicardial fat has been designated previously as brown-like fat. The supraclavicular fat depot in man has been defined as beige coexistent with classical brown based on its gene expression profile.

Objective: The aim of the study was to establish the gene expression profile and morphology of human epicardial and visceral paracardial fat compared with sc fat.

SAP97 controls the trafficking and resensitization of the beta-1-adrenergic receptor through its PDZ2 and I3 domains.

Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß1-adrenergic receptor (ß1-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß1-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß1-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß1-AR.

The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the β(1)-adrenergic receptor to G(s).

Abstract A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 159(3.53) to glutamic acid and isoleucine160(3.

Depot-specific overexpression of proinflammatory, redox, endothelial cell, and angiogenic genes in epicardial fat adjacent to severe stable coronary atherosclerosis.

Background: Pro- and antiinflammatory genes are expressed in epicardial adipose tissue (EAT). Our objectives were to characterize genes in EAT that may contribute specifically to coronary atherogenesis and to measure circulating adipokines matched to their messenger RNAs (mRNAs) in EAT. We hypothesized that severe coronary atherosclerosis (CAD) would preferentially affect gene expression in EAT as compared to substernal fat or subcutaneous thoracic adipose tissue (SAT), as well as circulating levels of adipokines.

Inflammatory genes in epicardial fat contiguous with coronary atherosclerosis in the metabolic syndrome and type 2 diabetes: changes associated with pioglitazone.

Objective: To determine changes in gene expression in epicardial adipose tissue (EAT) associated with coronary atherosclerosis (CAD) and effects of pioglitazone therapy.

Research Design And Methods: Genes were quantified by RT-PCR in EAT and thoracic subcutaneous adipose tissue (SAT) obtained during surgery in CAD patients with metabolic syndrome (MS) or type 2 diabetes and control subjects with minimal or no CAD and no MS or type 2 diabetes.

Results: Increased expression of interleukin-1 receptor antagonist (IL-1Ra) and IL-10, a trend for higher IL-1β, and no change in peroxisome proliferator-activated receptor-γ (PPARγ) was found in EAT from MS or type 2 diabetes.

Epicardial fat gene expression after aerobic exercise training in pigs with coronary atherosclerosis: relationship to visceral and subcutaneous fat.

Epicardial adipose tissue (EAT) is contiguous with coronary arteries and myocardium and potentially may play a role in coronary atherosclerosis (CAD). Exercise is known to improve cardiovascular disease risk factors. The purpose of this study was to investigate the effect of aerobic exercise training on the expression of 18 genes, measured by RT-PCR and selected for their role in chronic inflammation, oxidative stress, and adipocyte metabolism, in peri-coronary epicardial (cEAT), peri-myocardial epicardial (mEAT), visceral abdominal (VAT), and subcutaneous (SAT) adipose tissues from a castrate male pig model of familial hypercholesterolemia with CAD.

Rab11a and its binding partners regulate the recycling of the ß1-adrenergic receptor.

ß1-adrenergic receptors (ß1-AR) are internalized in response to agonists and then recycle back for another round of signaling. The serine 312 to alanine mutant of the ß1-AR (S312A) is internalized but does not recycle. We determined that WT ß1-AR and S312A were internalized initially to an early sorting compartment because they colocalized by >70% with the early endosomal markers rab5a and early endosomal antigen-1 (EEA1).

Human epicardial adipokine messenger RNAs: comparisons of their expression in substernal, subcutaneous, and omental fat.

We compared the gene expression of inflammatory and other proteins by real-time quantitative polymerase chain reaction in epicardial, substernal (mediastinal) and subcutaneous sternal, upper abdominal, and leg fat from coronary bypass patients and omental (visceral) fat from extremely obese women undergoing bariatric surgery. We hypothesized that (1) epicardial fat would exhibit higher expression of inflammatory messenger RNAs (mRNAs) than substernal and subcutaneous fat and (2) epicardial mRNAs would be similar to those in omental fat. Epicardial fat was clearly different from substernal fat because there was a far higher expression of haptoglobin, prostaglandin D(2) synthase, nerve growth factor beta, the soluble vascular endothelial growth factor receptor (FLT1), and alpha1 glycoprotein but not of inflammatory adipokines such as monocyte chemoattractant protein-1, interleukin (IL)-8, IL-1beta, tumor necrosis factor alpha, serum amyloid A, plasminogen activator inhibitor-1, or adiponectin despite underlying coronary atherosclerosis.

Compartmentalized cyclic adenosine 3',5'-monophosphate at the plasma membrane clusters PDE3A and cystic fibrosis transmembrane conductance regulator into microdomains.

Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function.

Uncoupling protein-1 and related messenger ribonucleic acids in human epicardial and other adipose tissues: epicardial fat functioning as brown fat.

Context: Uncoupling protein-1 (UCP-1) is the inner mitochondrial membrane protein that is a specific marker for and mediator of nonshivering thermogenesis in brown adipocytes.

Objective: This study was performed to better understand the putative thermogenic function of human epicardial fat.

Design: We measured the expression of UCP-1 and brown adipocyte differentiation transcription factors PR-domain-missing 16 (PRDM16) and peroxisome-proliferator-activated receptor gamma co-activator-1 alpha (PGC-1 alpha) in epicardial, substernal, and sc thoracic, abdominal, and leg fat.

Comparison of messenger RNA distribution for 60 proteins in fat cells vs the nonfat cells of human omental adipose tissue.

The messenger RNA (mRNA) distribution of 60 proteins was examined in the 3 fractions obtained by collagenase digestion (fat cells and the nonfat cells comprising the tissue remaining after collagenase digestion [matrix] and the stromovascular cells) of omental adipose tissue obtained from morbidly obese women undergoing bariatric surgery. Fat cells were enriched by at least 3-fold as compared with nonfat cells in the mRNAs for retinol binding protein 4, angiotensinogen, adipsin, glutathione peroxidase 3, uncoupling protein 2, peroxisome proliferator-activated receptor gamma, cell death-inducing DFFA-like effector A, fat-specific protein 27, 11beta-hydroxysteroid dehydrogenase 1, glycerol channel aquaporin 7, NADPH:quinone oxidoreductase 1, cyclic adenosine monophosphate phosphodiesterase 3B, glyceraldehyde-3-phosphate dehydrogenase, insulin receptor, and amyloid A1. Fat cells were also enriched by at least 26-fold in the mRNAs for proteins involved in lipolysis such as hormone-sensitive lipase, lipoprotein lipase, adipose tissue triglyceride lipase, and FAT/CD36.

Assembly of an SAP97-AKAP79-cAMP-dependent protein kinase scaffold at the type 1 PSD-95/DLG/ZO1 motif of the human beta(1)-adrenergic receptor generates a receptosome involved in receptor recycling and networking.

Appropriate trafficking of the beta(1)-adrenergic receptor (beta(1)-AR) after agonist-promoted internalization is crucial for the resensitization of its signaling pathway. Efficient recycling of the beta(1)-AR required the binding of the protein kinase A anchoring protein-79 (AKAP79) to the carboxyl terminus of the beta(1)-AR (Gardner, L. A.

AKAP79-mediated targeting of the cyclic AMP-dependent protein kinase to the beta1-adrenergic receptor promotes recycling and functional resensitization of the receptor.

Resensitization of G protein-coupled receptors (GPCR) following prolonged agonist exposure is critical for restoring the responsiveness of the receptor to subsequent challenges by agonist. The 3'-5' cyclic AMP-dependent protein kinase (PKA) and serine 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) were both necessary for efficient recycling and resensitization of the agonist-internalized beta(1)-AR (Gardner, L. A.