Differential expression pattern of CEACAM1 isoforms in polarized epithelial cells : it's regulation and some functional consequences
Author: Sundberg, Ulla
Date: 2004-02-06
Location: CMB:s auditorium, Institutionen för Cell och Molekylärbiologi, Berzelius väg 21, Karolinska Institutet
Time: 9.00
Department: Institutionen för cell- och molekylärbiologi (CMB) / Department of Cell and Molecular Biology
Abstract
The cell adhesion molecule CEACAM1 is expressed in epithelia, small
vessel endothelia and leukocytes as two major, co-expressed isoforms,
CEACAM1-L and CEACAM1-S, which differ in their cytoplasmic domains.
CEACAM1 has been found both in cell-cell contact areas and on apical
surfaces of polarized epithelial cells.
The aim of this project was to find out whether CEACAM1-L and CEACAM1S exhibit different surface localization in polarized epithelial cells, and if so to map the signals for the differential surface localization. MDCK cells were used as a model system and were transfected with wildtype and mutant forms of both CEACAM1 -L and CEACAM1-S.
We demonstrated that CEACAM1-L was expressed both on the apical and the lateral surfaces of polarized MDCK cells, whereas CEACAM1-S only appeared on the apical surface. We also showed that CEACAM1-L acts as a cell adhesion molecule in the lateral surfaces, binding adjacent cells together by a trans- homophilic complex formation. By double staining with both monoclonal and polyclonal antibodies we could demonstrate that CEACAM1-L occurred in two different conformational states, corresponding to the transhomophilic binding state and a nonbinding state. In CEACAM1-S the trans-homophilic binding state dominated.
We found that tyr515 has a crucial role for the lateral localization of CEACAM1-L and that the lysine residues 519-521 as well as ser503 also are important for the steady-state lateral localization. We also demonstrated that both pervanadate, which increases tyrosine phosphorylation, and staurosporine, which decreases serine phosphorylation, caused disappearance of CEACAM1-L from the lateral cell surfaces. By double staining for adaptor proteins AP-1 and AP-2 as well as for markers for early and late endosomes and lysosomes we obtained results suggesting that staurosporine stimulates internalization of CEACAM1-L from the plasma membrane to early and late endosomes and lysosomes, and that pervanadate causes an increased routing of CEACAM1-L from the trans-golgi network to late endosomes and lysosomes. We found no co-localization with the tight junction proteins occludin or ZO-1, but a prominent co-localization of CEACAM1-L with E-cadherin and beta-catenin in the adherens junctions. CEACAM1 -L did not co-localize with desmosomes. However, expression of CEACAM1-L caused a prominent reduction of the amount of desmosomal plaques in the lateral surfaces and a concomitant disorganization of the submembraneous cytokeratin filaments. The signal for desmosomal perturbation was mapped to the C-terminal part of the cytoplasmic domain of CEACAM1.
Last we studied the appearance of the two CEACAM1 isoforms in intracellular vesicles and primary cilia. CEACAM1-L but not CEACAM1-S, was found in prominent large tubulovacuolar structures in the subapical part of polarized MDCK cells. CEACAM1 -S was present in small vesicular structures. When the cells became superconfluent they develop primary cilia, that were identified by staining for alpha-tubulin and gamma-tubulin. Both CEACAM1-L and CEACAM1-S, were expressed on the cilia. Especially CEACAM1-S, was localized to the distal ends of the cilia. The distal ends of primary cilia from different cells were often seen in contact with each other, suggesting that CEACAM1 -S mediated adhesion between primary cilia.
The aim of this project was to find out whether CEACAM1-L and CEACAM1S exhibit different surface localization in polarized epithelial cells, and if so to map the signals for the differential surface localization. MDCK cells were used as a model system and were transfected with wildtype and mutant forms of both CEACAM1 -L and CEACAM1-S.
We demonstrated that CEACAM1-L was expressed both on the apical and the lateral surfaces of polarized MDCK cells, whereas CEACAM1-S only appeared on the apical surface. We also showed that CEACAM1-L acts as a cell adhesion molecule in the lateral surfaces, binding adjacent cells together by a trans- homophilic complex formation. By double staining with both monoclonal and polyclonal antibodies we could demonstrate that CEACAM1-L occurred in two different conformational states, corresponding to the transhomophilic binding state and a nonbinding state. In CEACAM1-S the trans-homophilic binding state dominated.
We found that tyr515 has a crucial role for the lateral localization of CEACAM1-L and that the lysine residues 519-521 as well as ser503 also are important for the steady-state lateral localization. We also demonstrated that both pervanadate, which increases tyrosine phosphorylation, and staurosporine, which decreases serine phosphorylation, caused disappearance of CEACAM1-L from the lateral cell surfaces. By double staining for adaptor proteins AP-1 and AP-2 as well as for markers for early and late endosomes and lysosomes we obtained results suggesting that staurosporine stimulates internalization of CEACAM1-L from the plasma membrane to early and late endosomes and lysosomes, and that pervanadate causes an increased routing of CEACAM1-L from the trans-golgi network to late endosomes and lysosomes. We found no co-localization with the tight junction proteins occludin or ZO-1, but a prominent co-localization of CEACAM1-L with E-cadherin and beta-catenin in the adherens junctions. CEACAM1 -L did not co-localize with desmosomes. However, expression of CEACAM1-L caused a prominent reduction of the amount of desmosomal plaques in the lateral surfaces and a concomitant disorganization of the submembraneous cytokeratin filaments. The signal for desmosomal perturbation was mapped to the C-terminal part of the cytoplasmic domain of CEACAM1.
Last we studied the appearance of the two CEACAM1 isoforms in intracellular vesicles and primary cilia. CEACAM1-L but not CEACAM1-S, was found in prominent large tubulovacuolar structures in the subapical part of polarized MDCK cells. CEACAM1 -S was present in small vesicular structures. When the cells became superconfluent they develop primary cilia, that were identified by staining for alpha-tubulin and gamma-tubulin. Both CEACAM1-L and CEACAM1-S, were expressed on the cilia. Especially CEACAM1-S, was localized to the distal ends of the cilia. The distal ends of primary cilia from different cells were often seen in contact with each other, suggesting that CEACAM1 -S mediated adhesion between primary cilia.
List of papers:
I. Sundberg U, Obrink B (2002). "CEACAM1 isoforms with different cytoplasmic domains show different localization, organization and adhesive properties in polarized epithelial cells. " J Cell Sci 115(Pt 6): 1273-84
Pubmed
II. Sundberg U, Beauchemin N, Obrink B (2004). "The cytoplasmic domain of CEACAM1-L controls it´s lateral localization and the organization of desmosomes in polarized epithelial cells." J Cell Sci (In Print)
View record in Web of Science®
III. Sundberg U, Obrink B (2004). "Differential expression pattern of CEACAM1-L and CEACAM1-S in intracellular vesicles and the primary cilium of polarized MDCK cells." (Manuscript)
I. Sundberg U, Obrink B (2002). "CEACAM1 isoforms with different cytoplasmic domains show different localization, organization and adhesive properties in polarized epithelial cells. " J Cell Sci 115(Pt 6): 1273-84
Pubmed
II. Sundberg U, Beauchemin N, Obrink B (2004). "The cytoplasmic domain of CEACAM1-L controls it´s lateral localization and the organization of desmosomes in polarized epithelial cells." J Cell Sci (In Print)
View record in Web of Science®
III. Sundberg U, Obrink B (2004). "Differential expression pattern of CEACAM1-L and CEACAM1-S in intracellular vesicles and the primary cilium of polarized MDCK cells." (Manuscript)
Issue date: 2004-01-16
Publication year: 2004
ISBN: 91-7349-774-6
Statistics
Total Visits
Views | |
---|---|
Differential ...(legacy) | 254 |
Differential ... | 88 |
Total Visits Per Month
October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | April 2024 | |
---|---|---|---|---|---|---|---|
Differential ... | 1 | 1 | 0 | 0 | 0 | 0 | 0 |
Top country views
Views | |
---|---|
China | 56 |
United States | 52 |
Sweden | 44 |
Germany | 40 |
South Korea | 10 |
United Kingdom | 7 |
Finland | 6 |
Ireland | 6 |
Denmark | 3 |
Hong Kong | 3 |
Top cities views
Views | |
---|---|
Beijing | 27 |
Kiez | 15 |
Seoul | 10 |
Sunnyvale | 8 |
Dublin | 6 |
Tianjin | 6 |
Mountain View | 5 |
Ludwigshafen am Rhein | 4 |
Ashburn | 3 |
Ballerup | 3 |