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Both Intra- and Extracellular Ca2 Participate in the Regulation of the Lateral Diffusion of the PDGF-β2 Receptor
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
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2000 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 20, no 2, p. 119-127Article in journal (Refereed) Published
Abstract [en]

When the receptors for platelet-derived growth factor (PDGF) are activatedthey aggregate, become tyrosine-phosphorylated and elicit a cascade ofdown-stream signals, including mobilization of Ca2+ from intra- andextracellular stores. Receptor mobility in the plane of the membrane isa prerequisite for receptor aggregation and further signalling. Using humanforeskin fibroblasts (AG 1523) and fluorescence recovery afterphotobleaching (FRAP), we therefore assessed the lateral mobilitycharacteristics of PDGF-β2 receptors by their diffusioncoefficient (D), and fraction of mobile receptors (R). This was done oncells stimulated with either normal human serum (NHS) or PDGF underdifferent Ca2+-conditions.

The results suggest that both intra- and extracellular free Ca2+influence the mobility characteristics of the PDGF-β2receptor. Interestingly, the extracellular Ca2+ seems to imposegeneral restrictions on the mobility of receptors, since R increased whenextracellular Ca2+ was quenched with EGTA, whereas intracellularclamping of Ca2+ transients with MABTAM (BAPT/AM) primarily affectedD. When both intra- and extracellular Ca2+ were quenced, D remainedlow and R high, further supporting the proposition that they achievedistinct effects. Inhibition of tyrosine phosphorylation with Erbstatin,partly inhibited the NHS effects and released PDGF-induced receptorimmobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGFinduced changes in intracellular free [Ca2+]. In view of the presentdata it might have important effects on the state of the receptor in themembrane, for instance by regulating its lateral mobility, communicationwith other receptors and signalling functions in the membrane.

Place, publisher, year, edition, pages
2000. Vol. 20, no 2, p. 119-127
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25447DOI: 10.1023/A:1005519501194Local ID: 9893OAI: oai:DiVA.org:liu-25447DiVA, id: diva2:245776
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Dynamics and gene expression of growth factor receptors in human cultured skin cells: Effects of UV radiation and calcium on EGF- and PDGF-receptors
Open this publication in new window or tab >>Dynamics and gene expression of growth factor receptors in human cultured skin cells: Effects of UV radiation and calcium on EGF- and PDGF-receptors
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Growth factors transmit biological signals for the stimulation of cell growth and their stimulation may be involved in turnourigenesis. It is therefore of great importance to understand growth factor receptor reactions in response to stimuli such as calcium depletion or ultraviolet radiation, which normal human cells are invariably exposed to during their growth cycle. We have studied human skin cells i.e. fibroblasts, lceratinocytes and melanocytes and their growth factor receptor expression on the surface of cells, reactions in the plane of the cell membrane, intracellular trafficlcing, and gene expression after exposure to their ligand, serum, UVB radiation and calcium depletion. We have used Fluorescence recovery after photo bleaching (FRAP) to assess receptor characteristics in cell membranes, confocal laser scanning microscopy to visualize receptor internalization, Ratio imaging for calcium studies, Northern blot for detection of the gene for the epidermal growth factor receptor (EGF-R) and flow cytometry for cell surface receptor determination. Platelet-derived growth factor receptors (PDGF-R) and EGF-R were studied since they affect cell proliferation and viability.

Our results showed that the addition of PDGF increased receptor mobility characteristics in normal fibroblasts, also "starvation" of cells increased their receptor mobility. We could show that changes in both intra-and extracellular free Ca2+ influence the mobility characteristics of PDGF-ß2 receptors. We have demonstrated that the three celltypes display different basal EGF-R mobility characteristics. After UVB irradiation mobility characteristics increased in all cell types but with differences in diffusion coefficients or mobile fractions. Addition of antioxidant enzymes, catalase and superoxide dismutase, prior to UVB irradiated cells abolished the UY-induced receptor mobility changes. We have shown that a single physiologic dose ofUVB radiation alters the intracellular EGF-R distribution and intracellular transport in melanocytes. It also significantly alters the melanocyte phenotype. We were able to detect a constitutive EGF-R gene expression and showed that UVB-radiation induces a time-dependent induction in EGF-R mRNA in melanocytes. Human melanocytes express EGF-R on their cell surface and UVR induces time dependent changes in the number of receptors but the number of receptors does not correlate with the level of UV-induced EGF-R gene expression.

It is concluded that UV-radiation, growth factors and calcium, ubiquitous constituents of every day life, all having tremendous effects in vivo, also affect human cells in vitro in parameters studied that are of importance for proliferation, survival and tumourigenesis.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2001. p. 88
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 674
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25680 (URN)10056 (Local ID)91-7219-965-2 (ISBN)10056 (Archive number)10056 (OAI)
Public defence
2001-05-18, Administrationshusets Aula, Universitetssjukhuset, Linköping, 09:30 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-06Bibliographically approved

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Wasteson, ÅkeLoitto, VesaMagnusson, Karl-Eric

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