scholarly journals OR34-02 Somatic Transmembrane Domain Mutations of a Cell Adhesion Molecule, CADM1, Cause Primary Aldosteronism by Preventing Gap Junction Communication Between Adrenocortical Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Xilin Wu ◽  
Sumedha Garg ◽  
Claudia P Cabrera ◽  
Elena Azizan ◽  
Junhua Zhou ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Primary Aldosteronism ◽  
Cell Adhesion Molecule ◽  
Transmembrane Domain ◽  
Fold Increase ◽  
Protein Modelling ◽  
Dye Transfer ◽  
Aldosterone Secretion ◽  
H295r Cells

Abstract Primary Aldosteronism (PA) is the commonest curable cause of hypertension. Whole exome sequencing (WES) in 2011 and 2013 identified common somatic mutations in genes regulating membrane polarisation in 60–80% of aldosterone-producing adenomas (APA). We undertook WES on 39 consecutive APAs in search of further variants. 1 APA revealed a somatic mutation (Val380Asp) within the single transmembrane domain of Cell Adhesion Molecule 1 (CADM1). An adjacent mutation (Gly379Asp) was discovered on WES from a PA patient in Munich. Both short and long isoforms (442 & 453 residues) of wild-type (WT) and both mutant CADM1 genes were cloned into lentivirus vectors and each transduced into adrenocortical (H295R) cells to assess its effect on aldosterone secretion and other parameters. Previous studies in pancreatic islet cells suggested a role of CADM1 in regulating gap junction (GJ) communication. To assess this we microinjected single WT or mutant H295R cells with the GJ permeable dye calceinAM and counted the dye-positive cells after 1 hour. The effect of inhibiting or silencing GJs in H295R cells using peptide gap27 or a Dharmacon smartpool was assessed. H295R cells were also co-transfected with WT or mutant CADM1 and the GJ protein CX43, tagged with the mApple fluorophore. These were mixed with cells transfected with CX43-Venus, allowing confocal visualisation of GJ formation. Protein modelling was undertaken to determine whether Asp in the intramembranous domain changes angulation of CADM1. All mutant isoforms had consistently different effects, shown as a range compared to WT. Cells transduced with mutant CADM1 showed 3-6-fold increase in aldosterone secretion (p<0.01) and 10-20-fold increase in CYP11B2 expression (p<0.001) compared to WT. Dye transfer assays showed paucity of dye transfer between neighbouring mutant CADM1 cells, while calcein passed easily through GJs in WT cells. CX43 inhibition increased aldosterone secretion 2-fold (p<0.01), and CYP11B2 expression 3 to 8-fold (<0.001). Knock-down of GJ proteins increased aldosterone secretion 1.5-fold (p<0.01) and CYP11B2 expression 1.7-fold (p<0.001). Protein modelling showed mutations to increase the angle of ectodomains to cell membrane, from 49o in WT cells, to 62o and 90o in Gly379Asp and Val380Asp respectively; increasing inter-cell distance from 21.2nm to 24.7 and 27.9nm. Mixing of Venus and mApple-tagged CX43 transfected cells showed fewer intact GJ channels in cells co-transfected with mutant compared to WT CADM1 [mutant 42/291 (14.4%) VS WT 68/212 (32.1%) p<0.001]. The CADM1 mutations shows the importance of membrane proteins in aldosterone regulation to extend beyond ion channels and transporters. A key role may be to bring opposing CX43 hemichannels close enough to form GJ channels, permitting the oscillating Ca2+ currents which regulate aldosterone in intact adrenal slices.

Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development

Development ◽  
2002 ◽  
Vol 129 (16) ◽  
pp. 3839-3850 ◽  
Author(s):  
Estella Wong ◽  
Chunzhong Yang ◽  
Jun Wang ◽  
Danny Fuller ◽  
William F. Loomis ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Sorting ◽  
Cell Adhesion Molecule ◽  
Fold Increase ◽  
Wild Type ◽  
Cell Type ◽  
Aberrant Cell ◽  
Adhesion Sites ◽  
Cada Gene

The cadA gene in Dictyostelium encodes the Ca2+-dependent cell adhesion molecule DdCAD-1, which is expressed soon after the initiation of development. To investigate the biological role of DdCAD-1, the cadA gene was disrupted by homologous recombination. The cadA-null cells showed a 50% reduction in EDTA-sensitive cell adhesion. The remaining EDTA-sensitive adhesion sites were resistant to dissociation by anti-DdCAD-1 antibody, suggesting that they were distinct adhesion sites. Cells that lacked DdCAD-1 were able to complete development and form fruiting bodies. However, they displayed abnormal slug morphology and culmination was delayed by ∼6 hours. The yield of spores was reduced by ∼50%. The proportion of prestalk cells in cadA– slugs showed a 2.5-fold increase over the parental strain. When cadA– cells were transfected with pcotB::GFP to label prespore cells, aberrant cell-sorting patterns in slugs became apparent. When mutant prestalk cells were mixed with wild-type prespore cells, mutant prestalk cells were unable to return to the anterior position of chimeric slugs, suggesting defects in the sorting mechanism. The wild-type phenotype was restored when cadA– cells were transfected with a cadA-expression vector. These results indicate that, in addition to cell-cell adhesion, DdCAD-1 plays a role in cell type proportioning and pattern formation.

scholarly journals The Cell Adhesion Molecule DdCAD-1 in Dictyostelium Is Targeted to the Cell Surface by a Nonclassical Transport Pathway Involving Contractile Vacuoles

1997 ◽  
Vol 138 (4) ◽  
pp. 939-951 ◽  
Author(s):  
Hiromi Sesaki ◽  
Estella F.S. Wong ◽  
Chi-Hung Siu
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Transport Mechanism ◽  
Cell Adhesion Molecule ◽  
Transmembrane Domain ◽  
Specific Inhibitor ◽  
Transport Pathway ◽  
Contractile Vacuoles

DdCAD-1 is a 24-kD Ca2+-dependent cell– cell adhesion molecule that is expressed soon after the initiation of development in Dictyostelium cells. DdCAD-1 is present on the cell surface as well as in the cytosol. However, the deduced amino acid sequence of DdCAD-1 lacks a hydrophobic signal peptide or any predicted transmembrane domain, suggesting that it may be presented on the cell surface via a nonclassical transport mechanism. Here we report that DdCAD-1 is transported to the cell surface via contractile vacuoles, which are normally involved in osmoregulation. Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuoles. Proteolytic treatment of isolated contractile vacuoles degraded vacuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD-1 was present in the lumen. The use of hyperosmotic conditions that suppress contractile vacuole activity led to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the absence of cell cohesiveness. Shifting cells back to a hypotonic condition after hypertonic treatments induced a rapid increase in DdCAD-1–positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell membrane. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific inhibitor of vacuolar-type H+-ATPase and thus of the activity of contractile vacuoles, also inhibited the accumulation of DdCAD-1 on the cell surface. Furthermore, an in vitro reconstitution system was established, and isolated contractile vacuoles were shown to import soluble DdCAD-1 into their lumen in an ATP-stimulated manner. Taken together, these data provide the first evidence for a nonclassical protein transport mechanism that uses contractile vacuoles to target a soluble cytosolic protein to the cell surface.

scholarly journals Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylinositol-linked form of the neural cell adhesion molecule.

1987 ◽  
Vol 105 (6) ◽  
pp. 2489-2500 ◽  
Author(s):  
H T He ◽  
J Finne ◽  
C Goridis
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Neural Cell Adhesion Molecule ◽  
Transmembrane Domain ◽  
Extracellular Fluid ◽  
Neural Cell ◽  
Ionic Detergent ◽  
Neural Cell Adhesion

The neural cell adhesion molecule (N-CAM) of rodents comprises three distinct proteins of Mr 180,000, 140,000, and 120,000 (designated N-CAM-180, -140, and -120). They are expressed in different proportions by different tissues and cell types. but the individual contribution of each form to cell adhesion is presently unknown. Previous studies have shown that the two N-CAM species of higher relative molecular mass span the membrane whereas N-CAM-120 lacks a transmembrane domain and can be released from the cell surface by phosphatidylinositol-specific phospholipase C. In this report, we provided evidence that N-CAM-120 contained covalently bound phosphatidylinositol and studied N-CAM-120 from its biosynthesis to its membrane insertion and finally to its release from the cell surface. Evidence was presented showing that the lipid tail of N-CAM-120 contained ethanolamine as is the case for other lipid-linked molecules. The phospholipid anchor was attached to the protein during the first minutes after completion of the polypeptide chain. This process took place in the endoplasmic reticulum as judged from endoglycosidase H digestion experiments. Immediately after a 2-min pulse with [35S]methionine, we detected also a short-lived precursor that had not yet acquired the lipid tail. Pulse-chase studies established that N-CAM-120 was transported to the cell surface from which it was slowly released into the extracellular milieu. The molecules recovered in the incubation medium appeared to have lost all of their bound fatty acid but only around half of the ethanolamine. Upon fractionation of brain tissue, approximately 75% of N-CAM-120 was recovered with a membrane fraction and approximately 25% in a membrane-free supernatant. A small proportion (approximately 6%) was found to be resistant to extraction by non-ionic detergent. A major posttranslational modification of N-CAM is polysialylation. Our results showed that also N-CAM-120 was polysialylated in the young postnatal brain and released in this form from cultured cerebellar cells. The presence of N-CAM in a form that can be released from the cell surface and accumulates in the extracellular fluid suggests a novel mechanism by which N-CAM-mediated adhesion may be modulated.

scholarly journals Identification of a new isoform of cell—cell adhesion molecule 105 (C-CAM), C-CAM4: a secretory protein with only one Ig domain

1996 ◽  
Vol 315 (3) ◽  
pp. 799-806 ◽  
Author(s):  
Karen EARLEY ◽  
Weiping LUO ◽  
Yuhong QIU ◽  
Nancy L. THOMPSON ◽  
Janice CHOU ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Transmembrane Domain ◽  
Blot Hybridization ◽  
The Other ◽  
Rnase Protection ◽  
Calculated Molecular Mass ◽  
Ig Domain ◽  
Cell Cell

A series of Southern blot hybridization experiments using probes derived from different regions of the rat liver cell–cell adhesion molecule 105 (C-CAM) cDNA revealed the presence of a 9.6 kb EcoRI genomic fragment that seemed to encode a unique C-CAM isoform. An RNase protection study showed that this C-CAM transcript was expressed in placenta, spleen, lung and large intestine. In contrast, the other C-CAM isoforms, C-CAM1 and C-CAM2, are expressed in liver and small intestine. This result also suggests that the new isoform, which we named C-CAM4, was indeed encoded by a new C-CAM gene. A rat placenta cDNA library was then screened and the full-length cDNA coding for C-CAM4 was isolated. The deduced protein contained 142 amino acids and had a calculated molecular mass of 15 kDa. C-CAM4 was composed of a leader sequence and the first V-like Ig domain typical of C-CAM-family proteins. However, C-CAM4 lacked the C-like Ig domains, the transmembrane domain, and the cytoplasmic domain found in other C-CAM isoforms. Thus, C-CAM4 is different from the other known C-CAMs in that it is a secreted protein. We have previously shown that the first Ig domain of C-CAM1 is crucial for its adhesion function. The V-like Ig domain of C-CAM4 had 92% and 89% sequence identity with the corresponding regions of C-CAM1 and C-CAM2 respectively. Together these results suggest that C-CAM4 may play a role in regulating the function of other C-CAM family proteins.

622: Von Hippel-Lindau Inactivation Downregulates the Cell-Cell Adhesion Molecule E Cadherin in Renal Cancer Cells

2005 ◽  
Vol 173 (4S) ◽  
pp. 170-170
Author(s):  
Maxine G. Tran ◽  
Miguel A. Esteban ◽  
Peter D. Hill ◽  
Ashish Chandra ◽  
Tim S. O'Brien ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cancer Cells ◽  
Renal Cancer ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Von Hippel Lindau ◽  
E Cadherin ◽  
Cell Cell
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