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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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Localization of  α4β7, αLβ2, αMβ2, and chimeric integrins by immunoelectron microscopy. K562  transfectants were stained  for protein expression using  12-nm gold particles (arrows)  as described in Materials and  Methods. Wild-type α4β7  (identified using the anti-β7  antibody, Fib 504) was localized predominantly to microvilli of K562-α4β7 tranfectants (A). K562-αLβ2 (B  and C) and K562-αMβ2 (D)  were stained with antibodies  to αL (B) or β2 (C and D),  demonstrating that αLβ2 and  αMβ2 were expressed mostly  on the cell body. K562 cells  transfected with the chimeric  integrins α4β7(αLβ2c) (E)  and α4β7(αMβ2tc) (F) were  stained with Fib 504. These  chimeric integrins were found  predominantly on microvilli.  Photomicrographs are representative of integrin distribution on the 50–100 cells examined in each sample. Bar,  0.5 μm.
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Figure 5: Localization of α4β7, αLβ2, αMβ2, and chimeric integrins by immunoelectron microscopy. K562 transfectants were stained for protein expression using 12-nm gold particles (arrows) as described in Materials and Methods. Wild-type α4β7 (identified using the anti-β7 antibody, Fib 504) was localized predominantly to microvilli of K562-α4β7 tranfectants (A). K562-αLβ2 (B and C) and K562-αMβ2 (D) were stained with antibodies to αL (B) or β2 (C and D), demonstrating that αLβ2 and αMβ2 were expressed mostly on the cell body. K562 cells transfected with the chimeric integrins α4β7(αLβ2c) (E) and α4β7(αMβ2tc) (F) were stained with Fib 504. These chimeric integrins were found predominantly on microvilli. Photomicrographs are representative of integrin distribution on the 50–100 cells examined in each sample. Bar, 0.5 μm.

Mentions: Previous reports demonstrated that integrin α4β7 localizes primarily to microvilli of mouse TK-1 lymphoma cells, whereas integrins αLβ2 and αMβ2 localize primarily to the cell body of TK-1 cells and human neutrophils respectively (Erlandsen et al., 1993; Berlin et al., 1995). We began by using immunoelectron microscopy to determine whether these integrins would localize similarly in transfected K562 cells. K562 cells had numerous microvillous projections. α4β7 was found primarily on microvilli (Fig. 5 A). In contrast, αLβ2 and αMβ2 integrins were located primarily on the cell body (Fig. 5, B–D). Both α4β7(αLβ2c) and α4β7(αMβ2tc) localized to microvillous projections (Fig. 5, E and F). A quantitative analysis of the distributions of these integrins is shown in Table I. The α4Δβ7 construct was also concentrated on microvilli (not shown). These results indicate that the transmembrane and cytoplasmic domains are not responsible for the differential localization of α4β7 versus αLβ2 and αMβ2.


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)

Localization of  α4β7, αLβ2, αMβ2, and chimeric integrins by immunoelectron microscopy. K562  transfectants were stained  for protein expression using  12-nm gold particles (arrows)  as described in Materials and  Methods. Wild-type α4β7  (identified using the anti-β7  antibody, Fib 504) was localized predominantly to microvilli of K562-α4β7 tranfectants (A). K562-αLβ2 (B  and C) and K562-αMβ2 (D)  were stained with antibodies  to αL (B) or β2 (C and D),  demonstrating that αLβ2 and  αMβ2 were expressed mostly  on the cell body. K562 cells  transfected with the chimeric  integrins α4β7(αLβ2c) (E)  and α4β7(αMβ2tc) (F) were  stained with Fib 504. These  chimeric integrins were found  predominantly on microvilli.  Photomicrographs are representative of integrin distribution on the 50–100 cells examined in each sample. Bar,  0.5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139798&req=5

Figure 5: Localization of α4β7, αLβ2, αMβ2, and chimeric integrins by immunoelectron microscopy. K562 transfectants were stained for protein expression using 12-nm gold particles (arrows) as described in Materials and Methods. Wild-type α4β7 (identified using the anti-β7 antibody, Fib 504) was localized predominantly to microvilli of K562-α4β7 tranfectants (A). K562-αLβ2 (B and C) and K562-αMβ2 (D) were stained with antibodies to αL (B) or β2 (C and D), demonstrating that αLβ2 and αMβ2 were expressed mostly on the cell body. K562 cells transfected with the chimeric integrins α4β7(αLβ2c) (E) and α4β7(αMβ2tc) (F) were stained with Fib 504. These chimeric integrins were found predominantly on microvilli. Photomicrographs are representative of integrin distribution on the 50–100 cells examined in each sample. Bar, 0.5 μm.
Mentions: Previous reports demonstrated that integrin α4β7 localizes primarily to microvilli of mouse TK-1 lymphoma cells, whereas integrins αLβ2 and αMβ2 localize primarily to the cell body of TK-1 cells and human neutrophils respectively (Erlandsen et al., 1993; Berlin et al., 1995). We began by using immunoelectron microscopy to determine whether these integrins would localize similarly in transfected K562 cells. K562 cells had numerous microvillous projections. α4β7 was found primarily on microvilli (Fig. 5 A). In contrast, αLβ2 and αMβ2 integrins were located primarily on the cell body (Fig. 5, B–D). Both α4β7(αLβ2c) and α4β7(αMβ2tc) localized to microvillous projections (Fig. 5, E and F). A quantitative analysis of the distributions of these integrins is shown in Table I. The α4Δβ7 construct was also concentrated on microvilli (not shown). These results indicate that the transmembrane and cytoplasmic domains are not responsible for the differential localization of α4β7 versus αLβ2 and αMβ2.

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