The Role of the CPNKEKEC Sequence in the β2 Subunit I Domain in Regulation of Integrin αLβ2 (LFA-1)

Abstract

The αL I (inserted or interactive) domain of integrin αLβ2 undergoes conformational changes upon activation. Recent studies show that the isolated, activated αL I domain is sufficient for strong ligand binding, suggesting the β2 subunit to be only indirectly involved. It has been unclear whether the activity of the αL I domain is regulated by the β2 subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169–176) in the β2 I domain with a corresponding β3 sequence, or mutating Lys174 to Thr, constitutively activates αLβ2 binding to ICAM-1. These mutants do not require Mn2+ for ICAM-1 binding and are insensitive to the inhibitory effect of Ca2+. We have also localized a component of the mAb 24 epitope (a reporter of β2 integrin activation) in the CPNKEKEC sequence. Glu173 and Glu175 of the β2 I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon β2 integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the αL I domain did not eliminate the mAb 24 epitope. This confirms that the αL I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the β2 subunit I domain. These results suggest that the CPNKEKEC sequence of the β2 I domain is involved in regulating the αL I domain.

Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2

“…m24 is an activation-reporting mAb that binds to the βI domain (27,28). The epitope of m24 maps to residues in the βI specificity determining loop (Glu-175) and α1-helix (Arg-122) near the β I MIDAS across from the α-subunit interface (Fig.…”

Crucial Role of the Specificity-determining Loop of the Integrin β4 Subunit in the Binding of Cells to Laminin-5 and Outside-in Signal Transduction

“…SDL of the ␤ 3 integrin subunit sequence is critically involved in specifying ligand binding of integrin ␣ v ␤ 3 (8). Moreover, a series of eight residues (CPNKEKEC) within the SDL region of the ␤ 2 integrin subunit regulates binding of the ␣ L ␤ 2 heterodimer to its ligand (22).…”

Membrane-proximal α/β Stalk Interactions Differentially Regulate Integrin Activation

“…The other three are located in the ␤A domain of the ␤ subunit. Besides the metal ion-dependent adhesion site (MIDAS), which is essential for ligand binding (9,10), the ␤A domain possesses two additional sites designated ADMIDAS (adjacent to MI-DAS) and LIMBS (ligand-associated metal binding site), respectively (11). Whereas ADMIDAS is occupied by a cation regardless of the presence of bound ligand, MIDAS and LIMBS have been shown to bind Mn 2ϩ only in the presence of bound ligand (3,11).…”