QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bodeau, A. L. Articles by LaFlamme, S. E. Search for Related Content PubMed PubMed Citation Articles by Bodeau, A. L. Articles by LaFlamme, S. E.

A functional comparison of mutations in integrin ß cytoplasmic domains

effects on the regulation of tyrosine phosphorylation, cell spreading, cell attachment and ß1 integrin conformation

The Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA
^* Author for correspondence (e-mail: laflams{at}mail.amc.edu )

View larger version (25K): [in a new window]   Fig. 1. Clustering chimeric receptors expressing wild-type integrin ß cytoplasmic domains induces tyrosine phosphorylation of p130CAS and paxillin. (A) Amino acid sequences of the homologous ß1 and ß3 cytoplasmic domains, as well as the alternatively spliced form ß3B, expressed in the context of chimeric receptors containing the extracellular and transmembrane domains (TM) of the tac subunit of the IL-2 receptor (tac chimeras). The ß4 cytoplasmic domain (not shown) is considerably larger and shares no homology with other integrin ß cytoplasmic domains. (B) Transiently transfected normal human fibroblasts expressing chimeras containing either the ß1, ß3, ß3B or ß4 cytoplasmic domains, or expressing the control receptor lacking a cytoplasmic domain (CR), were incubated in clustering assays for 40 minutes. Lysates (10 µg/lane) were separated by SDS-PAGE, western blotted for phosphotyrosine (upper panel) and reprobed for p130CAS (lower panel). (C) In separate clustering experiments, p130CAS was immunoprecipitated from 200 µg of lysates prepared from human fibroblasts expressing either the control receptor (CR) or the tac-ß1 chimera. Immunoprecipitates (IP) and 10 µg of cell lysates (lys) were separated by SDS-PAGE, western blotted for phosphotyrosine (upper panel) and reprobed for p130CAS and FAK (lower panels). (D) Similarly, paxillin was immunoprecipitated from 300 µg of lysates prepared from REF52 cells expressing either the control receptor (CR) or the tac-ß1 chimera. The IP and 10 µg of cell lysates (lys) were separated by SDS-PAGE, western blotted for phosphotyrosine (upper panel) and reprobed for paxillin (lower panel). P130, p130CAS; pax, paxillin.

View larger version (44K): [in a new window]   Fig. 4. Differences in phosphotyrosine signaling by the ß1-787-789 (*/A) and ß3-751-753 (*/A) mutants. (A) Normal human fibroblasts expressing tac chimeras containing either the wild-type ß1 or ß3 cytoplasmic domain or the ß1-787-789 (*/A) or ß3-751-753 (*/A) mutant were incubated in clustering assays for 40 minutes or 90 minutes. Lysates (10 µg/sample) were analyzed by SDS-PAGE and blotted for phosphotyrosine (upper panel), and then reprobed for p130CAS (lower panel). Samples were loaded as indicated. (B) Samples from the total population of transfected cells used for the study in A were examined by flow cytometry for the surface expression of the tac subunit of the IL-2 receptor. (C,D) Human fibroblasts were transfected with tac chimeras containing either the wild-type or mutant tails as indicated. Tac chimeras were clustered on the cell surface for 40 minutes. After preparation of cell lysates, p130CAS was immunoprecipitated from 200 µg of protein in C, or paxillin was immunoprecipitated from 150 µg in D and then separated by SDS-PAGE, blotted for phosphotyrosine (upper panel) and reprobed for p130CAS or paxillin (lower panels). Samples were loaded as indicated above each lane. These experiments were performed twice and similar results were obtained. P130, p130CAS; pax, paxillin.

View larger version (40K): [in a new window]   Fig. 2. Summary of mutations in the ß1 and ß3 cytoplasmic domains. Highly conserved regions of the ß1 and ß3 integrin cytoplasmic domains shown in red were targeted for point mutations. In most cases, the indicated residue (*) has been changed to alanine. However, other residues have been substituted as indicated. Mutated residues in the ß1 and ß3 tails are shown in blue. All of the ß cytoplasmic domains were expressed in the context of tac chimeras. Also provided is a summary chart of the activity of the different tac-ß tail mutants in the different assays. + to ++++ indicates the degree of similarity of tac-ß tail chimera activity to the wild-type tac-ß chimera in the assay; — indicates that the particular tac-ß tail chimera had no measurable activity in the assay; ND, mutant not tested in the assay; #, data summarized from Mastrangelo et al., 1999b.

View larger version (43K): [in a new window]   Fig. 3. Tyrosine phosphorylation triggered by clustering the various ß cytoplasmic domain mutants. Normal human fibroblasts expressing tac chimeras containing the various ß cytoplasmic domain mutations (A-C, ß1 mutants; E, ß3 mutants) were incubated in clustering assays for 40 minutes. Lysates (10 µg/sample) were separated by SDS-PAGE, blotted for phosphotyrosine (upper panels) and reprobed for p130CAS (lower panels). The ß1 and ß3 mutants were loaded as indicated above each lane. (D) A quantitative comparison of the ability of different tac-ß tail mutants to trigger tyrosine phosphorylation. The data from three separate experiments is presented as the mean ± s.e.m. A quantitative comparison of the ability of the ß3 mutants to activate tyrosine phosphorylation has already been published (Tahiliani et al., 1997).

View larger version (24K): [in a new window]   Fig. 5. The effects of ß1 cytoplasmic domain mutants on the expression of the 9EG7 epitope and cell attachment. (A) To examine the effects on the expression of the 9EG7 epitope, human fibroblasts were transiently transfected with the control receptor or tac chimeras containing the wild-type or mutant ß1 cytoplasmic domains as indicated. The effects of expressing these tac chimeras on the expression of the 9EG7 epitope were determined by two-color flow cytometry. Cells expressing levels of the chimeras between 103 and 104 fluorescence units were analyzed. The expression of the 9EG7 epitope was calculated relative to the total expression of ß1 integrins at the cell surface, which was determined using mAb K20. Because 9EG7 expression is reduced on cells expressing tac-ß1, the data is presented as % inhibition in 9EG7 expression on cells expressing the tac-ß tail chimeras compared to 9EG7 expression on cells expressing the control receptor. The data represent the mean from three separate experiments ± s.e.m. (B) To examine the effects on cell attachment, human fibroblasts were transiently transfected with the control chimeric receptor, or tac chimeras containing wild-type or mutant ß1 cytoplasmic domains as indicated. To determine the effects of expressing these tac-ß1 chimeras on cell attachment to fibronectin, the levels of chimera expression on attached cells, unattached cells and the starting population of cells (cells prior to attachment) were determined by flow cytometry. The mean fluorescence intensity of attached and unattached cells was compared to the starting population and is expressed as the percent of the starting population. Only cells expressing tac chimeras were analyzed (101-104 fluorescence units). The data represent the mean from three separate experiments ± s.e.m.

View larger version (35K): [in a new window]   Fig. 6. The effect of various mutations on the ability of the ß1 and ß3 cytoplasmic domains to regulate cell spreading. (A,B) Human fibroblasts were transiently transfected with the control receptor, or tac chimeras containing wild-type or mutant ß1 and ß3 cytoplasmic domains as indicated. Cells adherent to fibronectin for 1 hour were analyzed for cell-surface expression of tac and cell area as described in Materials and Methods. The cell area for 100 randomly sampled positively transfected cells is plotted as a function of tac expression. The x axis is a linear scale of cell area from 0 to 4000 µm2; the y axis is a linear scale of arbitrary FITC fluorescence (tac expression) units defined by Image Pro-Plus from 0 to 1.0x105 (A) and from 0 to 4.0x104 (B). We found in using this assay that round cells that have not begun to spread have cell areas less than 600 µm2 (Berrier et al., 2000). The percentage of positively transfected cells that had areas less than 600 µm2 was calculated from three experiments and graphed as the mean ± s.e.m. (C) The stable CHO cell lines A5 and ETC12 were transfected with the control receptor or tac chimeras containing either the wild-type ß1 or the ß1-787 (V/A) mutant cytoplasmic domain as indicated. A5 cells adherent to fibrinogen (15 µg/ml; Fg) for 30 minutes or ETC12 cells adherent to fibronectin (10 µg/ml; Fn) for 2 hours were analyzed for cell-surface expression of tac and cell area as described above. The cell area for 100 randomly sampled positively transfected cells is plotted as a function of tac expression. The x axis is a linear scale of cell area from 0 to 2000 µm2; the y axis is a linear scale of FITC fluorescence (tac expression) from 0 to 5.0x104. A vertical line positioned at 600 µm2 indicates the separation of spread (right) and not spread (left) cells. The percentage of positively transfected cells that had areas less than 600 µm2 was calculated from three experiments and graphed as the mean ± s.e.m.