Sarcolemmal Membrane-associated Protein Research Articles

Deletion of Sarcolemmal Membrane-Associated Protein Isoform 3 (SLMAP3) in Cardiac Progenitors Delays Embryonic Growth of Myocardium without Affecting Hippo Pathway.

The slmap gene is alternatively spliced to generate many isoforms that are abundant in developing myocardium. The largest protein isoform SLMAP3 is ubiquitously expressed and has been linked to cardiomyopathy, Brugada syndrome and Hippo signaling. To examine any role in cardiogenesis, mice homozygous for floxed slmap allele were crossed with Nkx2.5-cre mice to nullify its expression in cardiac progenitors. Targeted deletion of the slmap gene resulted in the specific knockout (KO) of the SLMAP3 (~91 KDa) isoform without any changes in the expression of the SLMAP2 (~43 kDa) or the SLMAP1 (~35 kDa) isoforms which continued to accumulate to similar levels as seen in Wt embryonic hearts. The loss of SLMAP3 from cardiac progenitors resulted in decreased size of the developing embryonic hearts evident at E9.5 to E16.5 with four small chambers and significantly thinner left ventricles. The proliferative capacity assessed with the phosphorylation of histone 3 or with Ki67 in E12.5 hearts was not significantly altered due to SLMAP3 deficiency. The size of embryonic cardiomyocytes, marked with anti-Troponin C, revealed significantly smaller cells, but their hypertrophic response (AKT1 and MTOR1) was not significantly affected by the specific loss of SLMAP3 protein. Further, no changes in phosphorylation of MST1/2 or YAP were detected in SLMAP3-KO embryonic myocardium, ruling out any impact on Hippo signaling. Rat embryonic cardiomyocytes express the three SLMAP isoforms and their knockdown (KD) with sh-RNA, resulted in decreased proliferation and enhanced senescence but without any impact on Hippo signaling. Collectively, these data show that SLMAP is critical for normal cardiac development with potential for the various isoforms to serve compensatory roles. Our data imply novel mechanisms for SLMAP action in cardiac growth independent of Hippo signaling.

Glucagon-Like Peptide-1 Receptor Agonist Protects Against Diabetic Cardiomyopathy by Modulating microRNA-29b-3p/SLMAP.

Our aims were to investigate the pathogenesis of diabetic cardiomyopathy (DCM) and to explore the protective effect of glucagon-like peptide-1 receptor agonist (GLP-1RA) on DCM. After 12 weeks of treatment with exenatide-loaded microspheres, a long-acting GLP-1RA, in DCM mice, cardiac structure and function were evaluated by plasma B-type natriuretic peptide (BNP), echocardiography, H&E, oil red and Sirius staining. The expression of glucagon-like peptide-1 receptor in mouse heart tissue was determined by immunofluorescence staining. The label-free proteomic analysis of cardiac proteins was conducted among control, DCM and DM+GLP-1RA groups. Then, quantitative real-time PCR, Western blotting and dual-luciferase reporter assay were performed to verify the regulation of target protein by the upstream microRNA (miRNA). GLP-1RA treatment obviously improved serum BNP, myocardial fibrosis, lipid deposition of the myocardium and echocardiography parameters in DCM mice. Sarcolemmal membrane-associated protein (SLMAP) was one of 61 differentially expressed cardiac proteins found in three groups by proteomic analysis. Up-regulation of microRNA-29b-3p (miR-29b-3p) and down-regulation of SLMAP were found in the ventricular myocardium of GLP-1RA-treated DCM mice. SLMAP was a target of miR-29b-3p, while GLP-1RA regulated SLMAP expression through miR-29b-3p. Furthermore, inhibition of glucagon-like peptide-1 receptor (GLP-1R) in cardiomyocytes reversed the effects of GLP-1RA on miR-29b/SLMAP. SLMAP may play roles in the pathogenesis of DCM and may be a target of GLP-1RA in protecting against DCM. After binding to myocardial GLP-1R, GLP-1RA can regulate the expression of myocardial SLMAP through miR-29b-3p.

An association between the sarcolemmal membrane-associated protein gene and microvascular endothelial diabetic retinopathy in patients with type 2 diabetes mellitus: A preliminary case control study

Background and aimsDiabetic retinopathy (DR) is one of the most common microvascular diabetic complications. Sarcolemmal membrane-associated protein (SLMAP) has been implicated in playing a role in microvascular endothelial dysfunction. This study aimed to assess the significance of SLMAP rs17058639C > T gene polymorphism among patients with type 2 diabetes mellitus (T2DM) and its relevance to microvascular endothelial diabetic retinopathy. MethodsWe conducted this case-control study on 100 individuals divided into 60 participants with T2DM and 40 healthy controls. Patients with T2D were stratified into two groups: 40 patients with DR and 20 patients with diabetic non-retinopathy (DNR). Patients with T2DM were compared with age- and sex-matched healthy controls. Fundus examinations were conducted to detect microvascular endothelial changes. The polymorphism of SLMAP rs17058639C > T gene was identified by real-time polymerase chain reaction (RT-PCR) TaqMan allelic discrimination. ResultsPatients with DR have significantly increased glycated hemoglobin (HbA1c) compared to patients with DNR (P < 0.001). There was no statistically significant difference found between diabetic and control groups regarding the frequency of SLMAP rs17058639C > T genotypes. The homozygous CC genotype was the most common variant among patients with DR; however, the results did not reach statistical significance. ConclusionsDiabetic retinopathy is correlated with poor glycemic control, and SLMAP rs17058639C > T polymorphism was associated with microvascular endothelial DR in patients with T2DM, although further studies with a large sample size are needed to confirm our findings.

SLMAP-3 is downregulated in human dilated ventricles and its overexpression promotes cardiomyocyte response to adrenergic stimuli by increasing intracellular calcium.

Structural dilation of cardiomyocytes (CMs) imposes a decline in cardiac performance that precipitates cardiac failure and sudden death. Since membrane proteins are implicated in dilated cardiomyopathy and heart failure, we evaluated the expression of the sarcolemmal membrane-associated protein (SLMAP) in dilated cardiomyopathy and its effect on CM contraction. We found that all 3 SLMAP isoforms (SLMAP-1, -2, and -3) are expressed in CMs and are downregulated in human dilated ventricles. Knockdown of SLMAPs in cultured CMs transduced with recombinant adeno-associated viral particles releasing SLMAP-shRNA precipitated reduced spontaneous contractile rate that was not fully recovered in SLMAP-depleted CMs challenged with isoproterenol (ISO), thus phenotypically mimicking heart failure performance. Interestingly, the overexpression of the SLMAP-3 full-length isoform induced a positive chronotropic effect in CMs that was more pronounced in response to ISO insult (vs. ISO-treated naïve CMs). Confocal live imaging showed that H9c2 cardiac myoblasts overexpressing SLMAP-3 exhibit a higher intracellular calcium transient peak when treated with ISO (vs. ISO-treated cells carrying a control adeno-associated viral particle). Proteomics revealed that SLMAP-3 interacts with the regulator of CM contraction, striatin. Collectively, our data demonstrate that SLMAP-3 is a novel regulator of CM contraction rate and their response to adrenergic stimuli. Loss of SLMAPs phenotypically mimics cardiac failure and crystallizes SLMAPs as predictive of dilated cardiomyopathy and heart failure.

A role for Sarcolemmal Membrane‐Associated Protein (SLMAP) in mediating autophagy in endothelial cells through an AMPK‐dependent mechanism

ObjectiveSarcolemmal membrane associated protein (SLMAP) belongs to the superfamily of tail‐anchored membrane proteins and is a component of endoplasmic reticulum (ER)/sarcoplasmic reticulum as well as mitochondria, cell surface membranes and the nuclear envelope. It has been reported to be involved in various physiological functions mainly in, for example, the regulation of ion channels, membrane fusion and vesicle transport. Previously, we have shown that the up‐regulation of SLMAP protein in diabetic db/db and Tally Ho mice was linked to endothelial dysfunction; however, until now no mechanistic evidence has been provided as to how an up‐regulation of SLMAP disrupts endothelial function in type 2 diabetes mellitus. The primary objective for the current study was to elucidate the cellular pathway(s) whereby an overexpression of SLMAP in human umbilical vein endothelial cells (HUVECs) results in endothelial dysfunction.Materials and MethodsHUVECs were cultured in M199 medium (Sigma Aldrich, USA) supplemented with 15% FBS, heparin (0.1% of 100mg/ml), ECGS (0.01% of 100 mg/ml), 100 μg/mL penicillin, and 100 U/mL streptomycin, at 37°C in a 5% CO2—95% air‐humidified incubator. The 41kDa isoform of SLMAP was overexpressed in HUVECs using SLMAP Lentiviral particles designed for humans (Accession number: AF304450.1; Genecopoeia), and SLMAP was partially knocked using small hairpin RNA (shRNA) lentiviral particles for humans (Santa Cruz Biotechnology, Catalogue number: sc‐78464‐V). The procedures were performed according to the manufacturer's instructions using polybrene at a concentration of 5μg/ml. After overexpression, the HUVECs were treated with, or without, the AMP‐activated protein kinase activator (AMPK), AICAR, 500μM, for 24 h. Protein markers of apoptosis, oxidative stress and autophagy were evaluated by Western Blotting, FACS, DHE staining and immunofluorescence techniques respectively.ResultsThe ER chaperone protein, GRP78; the unfolded protein response (UPR) signaling proteins, such as IRE‐1α, PERK, XBP1s, cleaved caspase‐3, p‐JNK, Bcl‐XL; and markers of autophagy, such as LC3B‐II, Beclin‐1 were found to be significantly elevated (p&lt;0.05), while protein levels of p‐AMPKα (Thr172) and SERCA2 were significantly decreased (p&lt;0.05) in HUVECs overexpressed with SLMAP. Interestingly, treatment with the AMPK activator, AICAR, significantly (p&lt;0.05) reversed all of the changes in HUVECs overexpressed with SLMAP. Moreover, the partial knock down of SLMAP significantly reduced IRE‐1α, PERK, XBP1s, p‐JNK, Bcl‐XL, LC3B‐II, Beclin‐1 proteins level, and increased p‐AMPKα (Thr172) and SERCA2 proteins level in SLMAP shRNA‐transfected HUVECs.ConclusionCollectively, this study has demonstrated for the first time that changes in the expression of SLMAP protein (41kDa) play an important role in ER stress and autophagy in endothelial cells via an AMPK‐dependent mechanism.Support or Funding InformationThis abstract is part of the project funded by NPRP: 5‐149‐3‐040

Identification of diagnostic biomarkers for early detection of anthrax from human aortic endothelial cells

Bacillus anthracis, an endospore-forming, gram-positive bacterium, causes anthrax in livestock and humans. Anthrax spores are one of the most threatening biological weapons and were used in the 2001 anthrax attacks in the USA. However, tools for early diagnosis and prediction of prognosis of anthrax patients did not exist until now. Here, our research focused on identifying biomarkers secreted from human aortic endothelial cells (HAECs) that could be applied to monitor anthrax patients. Biomarker candidates were identified by analysing the secretomes of HAECs after treating anthrax toxins by nano-liquid chromatography electrospray ionisation tandem mass spectrometry (nano-LC-ESI-MS/MS). We identified three secreted biomarker candidates: lactotransferrin (LTF), pregnancy zone protein (PZP), and sarcolemmal membrane-associated protein (SLMAP); these were also confirmed by immunoblot analysis. The results suggested that these novel biomarkers from anthrax toxin-treated HAECs could be utilised as diagnostic biomarkers of anthraxin future serological testing.

Abstract 235: Sarcolemmal Membrane Associated Protein Isoform 1: a Unique Regulator of Glucose Uptake and Metabolism in the Myocardium

As one of the leading causes of heart disease, diabetes is a problem which needs a solution. Regulation of glucose uptake and metabolism within skeletal and cardiac muscle has proven capable of altering systemic glucose levels and impacting metabolism to potentially improve patient outcomes. Unfortunately, to date, very few muscle specific metabolic regulators are known which can allow us to achieve blood glucose uptake and metabolism. Sarcolemmal Membrane Associated Protein Isoform 1 (SLMAP1) is a novel protein expressed predominantly within muscle tissue. It has been linked to diabetes through animal models, although its role in metabolism remains to be defined. Here we describe a novel role for SLMAP1 in glucose metabolism within the myocardium. We engineered a transgenic (TG) mouse with cardiac specific expression of SLMAP1. Using neonatal cardiomyocytes (NCMs) collected from these mice we performed glucose uptake assays with 2-deoxy-glucose (2DG), measured glycolytic rate using an Extracellular Flux XF24e Bioanalyzer, and analyzed glucose transporter 4 (GLUT4) trafficking using Western Blots, qPCR, and immunofluorescence imaging. NCMs extracted from TG hearts expressing SLMAP1 displayed increased levels of 2DG uptake (93% ± 25%, n=5, P&lt;0.01), basal glycolysis (glycolysis (92 ± 40%, n=5, P&lt;0.05), and maximal glycolysis (75 ± 31%, n=5, P&lt;0.05) compared with wildtype littermates. Confocal microscopy revealed both increased localization of glucose transporter 4 (GLUT4) at the cell surface as well as an expansion of GLUT4 early endosomes in TG NCMs. The data here indicates SLMAP1 as a novel regulator of glucose uptake and metabolism in the myocardium. The targeted expression of SLMAP1 in a muscle specific manner may enhance systemic glycemic control and serve to limit cardiovascular disease in metabolic disorders such as diabetes.

A fungal sarcolemmal membrane-associated protein (SLMAP) homolog plays a fundamental role in development and localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria.

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.

Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

Pinellia pedatisecta agglutinin (PPA) has previously been used in labeling fractions of myeloid leukemia cells in our laboratory. We report here that a bacterial expressed recombinant PPA domain b tagged with soluble coxsackie and adenovirus receptor (sCAR-PPAb) preferentially recognized drug resistant cancer cells K562/ADR and H460/5Fu, as compared to their parental cell lines. Pretreatment of K562/ADR cells with sCAR-PPAb significantly enhanced phagocytosis of K562/ADR by macrophages in vivo. Meanwhile, in a K562/ADR xenograft model, intratumoral injection of sCAR-PPAb induced macrophage infiltration and phagocytosis. Furthermore, immunoprecipitation, mass spectrometry and Western blot identified the membrane target of PPA on K562/ADR as sarcolemmal membrane associated protein (SLMAP). An antibody against SLMAP significantly promoted the phagocytosis of K562/ADR by macrophages in vitro. These findings suggest that PPA not only could be developed into a novel agent that can detect drug resistant cancer cells and predict chemotherapy outcome, but also it has potential value in immunotherapy against drug resistant cancer cells through inducing the tumoricidal activity of macrophages.

A Novel Disease Gene for Brugada Syndrome

Background— Mutations in genes including SCN5A encoding the α-subunit of the cardiac sodium channel (hNav1.5) cause Brugada syndrome via altered function of cardiac ion channels, but more than two-thirds of Brugada syndrome remains pathogenetically elusive. T-tubules and sarcoplasmic reticulum are essential in excitation of cardiomyocytes, and sarcolemmal membrane-associated protein (SLMAP) is a protein of unknown function localizing at T-tubules and sarcoplasmic reticulum. Methods and Results— We analyzed 190 unrelated Brugada syndrome patients for mutations in SLMAP . Two missense mutations, Val269Ile and Glu710Ala, were found in heterozygous state in 2 patients but were not found in healthy individuals. Membrane surface expression of hNav1.5 in the transfected cells was affected by the mutations, and silencing of mutant SLMAP by small interfering RNA rescued the surface expression of hNav1.5. Whole-cell patch-clamp recordings of hNav1.5-expressing cells transfected with mutant SLMAP confirmed the reduced hNav1.5 current. Conclusions— The mutations in SLMAP may cause Brugada syndrome via modulating the intracellular trafficking of hNav1.5 channel.

Increased Expression of the Tail-Anchored Membrane Protein SLMAP in Adipose Tissue from Type 2Tally HoDiabetic Mice

The tail-anchored membrane protein, sarcolemmal membrane associated protein (SLMAP) is encoded to a single gene that maps to the chromosome 3p14 region and has also been reported in certain diabetic populations. Our previous studies with db/db mice shown that a deregulation of SLMAP expression plays an important role in type 2 diabetes. Male Tally Ho mice were bred to present with either normoglycemia (NG) or hyperglycemia (HG). Abdominal adipose tissue from male Tally Ho mice of the HG group was found to have a significantly lower expression of the membrane associated glucose transporter-4 (GLUT-4) and higher expression of SLMAP compared to tissue from NG mice. There were 3 isoforms expressed in the abdominal adipose tissue, but only 45 kDa isoform of SLMAP was associated with the GLUT-4 revealed by immunoprecipitation data. Knock down studies using SLMAP siRNA with adipocytes resulted in a significant reduction in SLMAP and a decrease in glucose uptake. Thus, SLMAP may be an important regulator of glucose uptake or involved in GLUT-4 fusion/translocation into the plasma membrane of mouse abdominal adipose tissue and changes in SLMAP expression are linked to hyperglycemia and diabetes.

Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting.

BackgroundTail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown.ResultsTA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1.ConclusionThus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

A PP2A Phosphatase High Density Interaction Network Identifies a Novel Striatin-interacting Phosphatase and Kinase Complex Linked to the Cerebral Cavernous Malformation 3 (CCM3) Protein

The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B''' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.

Expression of the tail‐anchored protein SLMAP in developing myocardium and during remodelling after myocardial infarction

The Organization of Excitation-Contraction (E-C) coupling mechanism is temporally regulated during heart remodelling. The molecular components involved in organizing membranes junctions with respect to the contractile apparatus remain elusive. SLMAP (Sarcolemmal Membrane Associated Protein) is a unique molecule shown to localize in cardiac membranes as well as the contractile apparatus and may serve to organizing the E-C coupling mechanism. The aim of this study was to investigate SLMAP expression and localization in developing myocardium and after Myocardial Infarction (MI) to correlate changes with any reorganization of the E-C coupling mechanism. SLMAP was located at the Z-disc, T-tubules and Sarcolemmal Membranes of rat myocardium as assessed by Immunostaining and Western blot analysis. SLMAP expression was significantly increased (p<0.005) from embryonic (E) day 16 to postnatal (P1) and gradually decreased (p<0.01) to low levels at 13 weeks. In a rat heart MI model induced by coronary ligation, SLMAP1 levels significantly increased (4 weeks post-surgery) in Infarct tissue vs. remote left ventricle (LV) (p<0.05) and SHAM (p<0.005). The location of SLMAP and temporal changes in its expression in normal and defective myocardium suggest that SLMAP may serve a role in organizing the E-C coupling apparatus during normal cardiac growth and in remodelling post MI. This work was supported by a grant from HSFO.

Endothelial dysfunction in Type 2 diabetes correlates with deregulated expression of the tail-anchored membrane protein SLMAP

The Type 2 diabetic db/db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/db mouse to the alpha1-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-gamma agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/db microvasculature, which were markedly downregulated during COOH treatment but elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the overexpression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes.

Molecular properties of cardiac tail-anchored membrane protein SLMAP are consistent with structural role in arrangement of excitation-contraction coupling apparatus

The spatial arrangement of the cell-surface membranes (sarcolemma and transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitation-contraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique COOH-terminal membrane anchors that could target this molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac-specific expression of SLMAP isoforms that can be targeted to distinct subcellular membranes, self assemble, and interact with the myofibril suggests a potential role for this molecule in the structural arrangement of the E-C coupling apparatus.

A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre.

The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with gamma-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.