The N-terminal region and the mid-region complex of the integrin beta 2 subunit

Suet Mien Tan, M K Robinson, K Drbal, Y van Kooyk, J M Shaw, S K Law

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of the beta(2) subunit were replaced by those of beta(7). Changing either the beta(2) NTR or mid-region, but not the I-like domain to that of beta(7) did not affect LFA-1 (alpha(L)beta(2)) formation and surface expression. Thus, the specificity of alpha(L)beta(2) pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-beta(2) mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the beta(2) NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the beta(2) NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.

Original languageEnglish
Pages (from-to)36370-6
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number39
DOIs
Publication statusPublished - 28 Sep 2001

Cite this

Tan, Suet Mien ; Robinson, M K ; Drbal, K ; van Kooyk, Y ; Shaw, J M ; Law, S K. / The N-terminal region and the mid-region complex of the integrin beta 2 subunit. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 39. pp. 36370-6.
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The N-terminal region and the mid-region complex of the integrin beta 2 subunit. / Tan, Suet Mien; Robinson, M K; Drbal, K; van Kooyk, Y; Shaw, J M; Law, S K.

In: Journal of Biological Chemistry, Vol. 276, No. 39, 28.09.2001, p. 36370-6.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - The N-terminal region and the mid-region complex of the integrin beta 2 subunit

AU - Tan, Suet Mien

AU - Robinson, M K

AU - Drbal, K

AU - van Kooyk, Y

AU - Shaw, J M

AU - Law, S K

PY - 2001/9/28

Y1 - 2001/9/28

N2 - In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of the beta(2) subunit were replaced by those of beta(7). Changing either the beta(2) NTR or mid-region, but not the I-like domain to that of beta(7) did not affect LFA-1 (alpha(L)beta(2)) formation and surface expression. Thus, the specificity of alpha(L)beta(2) pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-beta(2) mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the beta(2) NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the beta(2) NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.

AB - In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of the beta(2) subunit were replaced by those of beta(7). Changing either the beta(2) NTR or mid-region, but not the I-like domain to that of beta(7) did not affect LFA-1 (alpha(L)beta(2)) formation and surface expression. Thus, the specificity of alpha(L)beta(2) pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-beta(2) mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the beta(2) NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the beta(2) NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.

KW - Amino Acid Sequence

KW - Animals

KW - Antibodies, Monoclonal/chemistry

KW - CD18 Antigens/chemistry

KW - COS Cells

KW - Cell Adhesion

KW - DNA, Complementary/metabolism

KW - Epitopes

KW - Flow Cytometry

KW - Gene Library

KW - Humans

KW - Intercellular Adhesion Molecule-1/metabolism

KW - Lymphocyte Function-Associated Antigen-1/metabolism

KW - Molecular Sequence Data

KW - Precipitin Tests

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Transfection

U2 - 10.1074/jbc.M102392200

DO - 10.1074/jbc.M102392200

M3 - Article

VL - 276

SP - 36370

EP - 36376

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 39

ER -