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Lateral diffusion of PDGF β-receptors in human fibroblasts
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.
1991 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 11, no 1, 43-52 p.Article in journal (Refereed) Published
Abstract [en]

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum of PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding 'normal' and 'starved' cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0 x 10-10 cm2s-1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor. and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

Place, publisher, year, edition, pages
1991. Vol. 11, no 1, 43-52 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-81072DOI: 10.1007/BF01118604OAI: oai:DiVA.org:liu-81072DiVA: diva2:550185
Available from: 2012-09-06 Created: 2012-09-06 Last updated: 2017-12-07Bibliographically 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. 88 p.
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, ÅkeMagnusson, Karl-Eric

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