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Presentation of integrins on leukocyte microvilli: a role for the extracellular domain in determining membrane localization.

Abitorabi MA, Pachynski RK, Ferrando RE, Tidswell M, Erle DJ - J. Cell Biol. (1997)

Bottom Line: Therefore, differences between the transmembrane and cytoplasmic domains of alpha4 and beta2 integrins do not account for differences in ability to support attachment under flow or in membrane localization.Integrins alpha4beta1, alpha5beta1, alpha6Abeta1, alphavbeta3, and alphaEbeta7 also localized to microvilli.Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well.

View Article: PubMed Central - PubMed

Affiliation: The Lung Biology Center, Department of Medicine, University of California, San Francisco, California 94143, USA.

ABSTRACT
Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins alpha4beta7, alphaLbeta2, and alphaMbeta2 were heterologously expressed in K562 cells. alpha4beta7 initiated adhesion under flow and localized to microvilli, whereas beta2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the alpha4beta7 cytoplasmic and/or transmembrane domains with the homologous domains of alphaLbeta2 or alphaMbeta2. Unexpectedly, these chimeras efficiently mediated adhesion to the alpha4beta7 ligand mucosal addressin cell adhesion molecule-1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of alpha4 and beta2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins alpha4beta1, alpha5beta1, alpha6Abeta1, alphavbeta3, and alphaEbeta7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.

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Cell surface expression of wild-type and chimeric integrins. K562-α4β7 (A), K562-α4β7 (αLβ2c) (B), and K562-α4β7  (αMβ2tc) (C) cells were stained with the anti-β7 antibody, Fib  504, as shown. Each of these three transfectants was also recognized by other antibodies specific for the α4 subunit or the α4β7  heterodimer, but were not recognized by anti-αE antibodies (not  shown). K562-αLβ2 were recognized by antibodies to β2 (D) and  αL (not shown). K562-αMβ2 cells were recognized by antibodies  to β2 (E) and αM (not shown). K562-αEβ7 cells stained with antibodies to β7 (F) and αE, but not with anti-α4 antibodies (not  shown). K562-α6Aβ1 cells were stained with the anti-α6 antibody  GoH3 (G). There was low level expression of αv on nontransfected K562 cells, and higher expression on K562-αvβ3 cells as  determined using the anti-αv antibody L230 (H). Fluorescence  intensity is shown on a log scale (one log per division). Dotted  and solid histograms represent staining with nontransfected and  transfected K562 cells, respectively.
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Figure 2: Cell surface expression of wild-type and chimeric integrins. K562-α4β7 (A), K562-α4β7 (αLβ2c) (B), and K562-α4β7 (αMβ2tc) (C) cells were stained with the anti-β7 antibody, Fib 504, as shown. Each of these three transfectants was also recognized by other antibodies specific for the α4 subunit or the α4β7 heterodimer, but were not recognized by anti-αE antibodies (not shown). K562-αLβ2 were recognized by antibodies to β2 (D) and αL (not shown). K562-αMβ2 cells were recognized by antibodies to β2 (E) and αM (not shown). K562-αEβ7 cells stained with antibodies to β7 (F) and αE, but not with anti-α4 antibodies (not shown). K562-α6Aβ1 cells were stained with the anti-α6 antibody GoH3 (G). There was low level expression of αv on nontransfected K562 cells, and higher expression on K562-αvβ3 cells as determined using the anti-αv antibody L230 (H). Fluorescence intensity is shown on a log scale (one log per division). Dotted and solid histograms represent staining with nontransfected and transfected K562 cells, respectively.

Mentions: To examine the role of cytoplasmic and transmembrane domains of α4β7 in adhesion and membrane localization, we expressed chimeric integrin heterodimers (Fig. 1). Each construct included the extracellular domains of α4 and β7. In one construct, designated α4β7(αLβ2c), most of the cytoplasmic domains of α4 and β7 were replaced with homologous regions of αL and β2. In a second construct, α4β7(αMβ2tc), all of the transmembrane and cytoplasmic domains of α4 and β7 were replaced with homologous domains of αM and β2. The integrin α and β subunit cDNAs were cotransfected into K562 human erythroleukemia cells, which do not normally express α4, αL, αM, β2, or β7. The levels of protein expression on the transfectants K562-α4β7, K562-α4β7(αLβ2c), and K562-α4β7(αMβ2tc) were determined to be similar by flow cytometry (Fig. 2, A–C). Two truncated cDNAs, α4Δ (truncated after amino acids GFFKR) and β7Δ (truncated after amino acids VLAYR), were also produced. The α4Δ was expressed in combination with β7 on transfected K562 cells (K562-α4Δβ7), although at levels somewhat below those seen with other constructs (data not shown). We were unable to detect expression of β7Δ on cells cotransfected with α4, despite a previous report that the homologous truncation mutant of mouse β7 was expressed on transfected cells (Crowe et al., 1994). We were able to document heterologous expression of other wild-type integrins, including αLβ2, αMβ2, α6Aβ1, αvβ3, and αEβ7, on appropriate K562 transfectants by flow cytometry (Fig. 2, D–H).


Presentation of integrins on leukocyte microvilli: a role for the extracellular domain in determining membrane localization.

Abitorabi MA, Pachynski RK, Ferrando RE, Tidswell M, Erle DJ - J. Cell Biol. (1997)

Cell surface expression of wild-type and chimeric integrins. K562-α4β7 (A), K562-α4β7 (αLβ2c) (B), and K562-α4β7  (αMβ2tc) (C) cells were stained with the anti-β7 antibody, Fib  504, as shown. Each of these three transfectants was also recognized by other antibodies specific for the α4 subunit or the α4β7  heterodimer, but were not recognized by anti-αE antibodies (not  shown). K562-αLβ2 were recognized by antibodies to β2 (D) and  αL (not shown). K562-αMβ2 cells were recognized by antibodies  to β2 (E) and αM (not shown). K562-αEβ7 cells stained with antibodies to β7 (F) and αE, but not with anti-α4 antibodies (not  shown). K562-α6Aβ1 cells were stained with the anti-α6 antibody  GoH3 (G). There was low level expression of αv on nontransfected K562 cells, and higher expression on K562-αvβ3 cells as  determined using the anti-αv antibody L230 (H). Fluorescence  intensity is shown on a log scale (one log per division). Dotted  and solid histograms represent staining with nontransfected and  transfected K562 cells, respectively.
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Related In: Results  -  Collection

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Figure 2: Cell surface expression of wild-type and chimeric integrins. K562-α4β7 (A), K562-α4β7 (αLβ2c) (B), and K562-α4β7 (αMβ2tc) (C) cells were stained with the anti-β7 antibody, Fib 504, as shown. Each of these three transfectants was also recognized by other antibodies specific for the α4 subunit or the α4β7 heterodimer, but were not recognized by anti-αE antibodies (not shown). K562-αLβ2 were recognized by antibodies to β2 (D) and αL (not shown). K562-αMβ2 cells were recognized by antibodies to β2 (E) and αM (not shown). K562-αEβ7 cells stained with antibodies to β7 (F) and αE, but not with anti-α4 antibodies (not shown). K562-α6Aβ1 cells were stained with the anti-α6 antibody GoH3 (G). There was low level expression of αv on nontransfected K562 cells, and higher expression on K562-αvβ3 cells as determined using the anti-αv antibody L230 (H). Fluorescence intensity is shown on a log scale (one log per division). Dotted and solid histograms represent staining with nontransfected and transfected K562 cells, respectively.
Mentions: To examine the role of cytoplasmic and transmembrane domains of α4β7 in adhesion and membrane localization, we expressed chimeric integrin heterodimers (Fig. 1). Each construct included the extracellular domains of α4 and β7. In one construct, designated α4β7(αLβ2c), most of the cytoplasmic domains of α4 and β7 were replaced with homologous regions of αL and β2. In a second construct, α4β7(αMβ2tc), all of the transmembrane and cytoplasmic domains of α4 and β7 were replaced with homologous domains of αM and β2. The integrin α and β subunit cDNAs were cotransfected into K562 human erythroleukemia cells, which do not normally express α4, αL, αM, β2, or β7. The levels of protein expression on the transfectants K562-α4β7, K562-α4β7(αLβ2c), and K562-α4β7(αMβ2tc) were determined to be similar by flow cytometry (Fig. 2, A–C). Two truncated cDNAs, α4Δ (truncated after amino acids GFFKR) and β7Δ (truncated after amino acids VLAYR), were also produced. The α4Δ was expressed in combination with β7 on transfected K562 cells (K562-α4Δβ7), although at levels somewhat below those seen with other constructs (data not shown). We were unable to detect expression of β7Δ on cells cotransfected with α4, despite a previous report that the homologous truncation mutant of mouse β7 was expressed on transfected cells (Crowe et al., 1994). We were able to document heterologous expression of other wild-type integrins, including αLβ2, αMβ2, α6Aβ1, αvβ3, and αEβ7, on appropriate K562 transfectants by flow cytometry (Fig. 2, D–H).

Bottom Line: Therefore, differences between the transmembrane and cytoplasmic domains of alpha4 and beta2 integrins do not account for differences in ability to support attachment under flow or in membrane localization.Integrins alpha4beta1, alpha5beta1, alpha6Abeta1, alphavbeta3, and alphaEbeta7 also localized to microvilli.Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well.

View Article: PubMed Central - PubMed

Affiliation: The Lung Biology Center, Department of Medicine, University of California, San Francisco, California 94143, USA.

ABSTRACT
Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins alpha4beta7, alphaLbeta2, and alphaMbeta2 were heterologously expressed in K562 cells. alpha4beta7 initiated adhesion under flow and localized to microvilli, whereas beta2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the alpha4beta7 cytoplasmic and/or transmembrane domains with the homologous domains of alphaLbeta2 or alphaMbeta2. Unexpectedly, these chimeras efficiently mediated adhesion to the alpha4beta7 ligand mucosal addressin cell adhesion molecule-1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of alpha4 and beta2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins alpha4beta1, alpha5beta1, alpha6Abeta1, alphavbeta3, and alphaEbeta7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.

Show MeSH
Related in: MedlinePlus