Mutation of a Conserved Asparagine in the I-like Domain Promotes Constitutively Active Integrins αLβ2 and αIIbβ3

Ming Cheng,Shen-Yun Foo,Min-Long Shi,Ren-Hong Tang,Le-Sheng Kong,S.K Alex Law,Suet-Mien Tan

doi:10.1074/jbc.m701386200

Abstract

The leukocyte beta2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of beta2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the beta2 inserted (I)-like domain. This mutation converted alphaLbeta2 from a resting into a high affinity conformer because alphaLbeta2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by alphaLbeta2N329S because of its reactivity with the beta2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation in beta3 generated constitutively active alphaIIbbeta3 that adhered to fibrinogen. This Asn is conserved in all human beta subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of the alpha7 helix and the beta6-alpha7 loop in the I-like domain.

Highlights

Introduction ofN339S in ␤3, Which Corresponds to N329S in ␤2, Generates a Constitutively Active I-less Integrin ␣IIb␤3—The Asn at position 329 of integrin ␤2 is conserved in all integrin ␤ subunits (Fig. 1A)
We showed previously that an intermediate affinity ␣L␤2 adhered to intercellular adhesion molecule (ICAM)-1 but that a high affinity conformer was required for adhesion to ICAM-3 [22]
␣L␤2N329S transfectant showed a high level of constitutive adhesion to ICAM-1 even in the absence of activating agent, whereas wild-type ␣L␤2 required activation with Mg/EGTA (Fig. 2)

Summary

Introduction

Introduction ofN339S in ␤3 , Which Corresponds to N329S in ␤2 , Generates a Constitutively Active I-less Integrin ␣IIb␤3—The Asn at position 329 of integrin ␤2 is conserved in all integrin ␤ subunits (Fig. 1A). The I-like domain mutations S116P and D209H generate ␤2 integrins that were expressed on the cell surface but dysfunctional [18, 19]. ␣L␤2N329S transfectant showed a high level of constitutive adhesion to ICAM-1 even in the absence of activating agent, whereas wild-type ␣L␤2 required activation with Mg/EGTA (Fig. 2).

Results

Conclusion