Abstract
Integrins are α/β heterodimeric cell adhesion receptors with each subunit comprising of a large extracellular domain, a single-spanning transmembrane domain, and usually a short cytoplasmic tail. Different combinations of 18 α and 8 β subunits make up 24 integrin members that recognize diverse extracellular ligands important in numerous biological functions such as immune responses, maintenance of hemostasis, and development. Abnormal activation of integrin is associated with many pathological conditions including thrombosis, inflammatory diseases, as well as tumor-driven cell growth, metastasis, and angiogenesis. Therefore, tight regulation is crucial in integrin activation. Recent structural and functional studies have shown that integrin activation is regulated by the cytoplasmic tails. Studies on the mechanism of integrin activation from inside the cell (namely inside-out activation) have been focused on the β cytoplasmic tail that is relatively conserved and bears binding sites for the common intracellular activators such as talin and kindlin. However, the role of α cytoplasmic tail in integrin activation remains elusive. The integrin α cytoplasmic tails share a conserved GFFKR motif at the membrane-proximal region that forms a specific interface with the membrane-proximal region of the β cytoplasmic tail. In contrast, the membrane-distal (MD) regions following the GFFKR motif are diverse significantly both in length, sequence and structure when reported, and their roles in integrin activation have not been well characterized. Our recent studies demonstrated that the α-MD region is required for talin and kindlin-induced activation of αIIb, αV, and αL integrins and suggest that the sequence diversity of the α-MD region might play a role in the regulation of integrin activation. In this study, we further examined the role of α-MD regions in integrin inside-out activation using αIIb, αL, and α5 integrins as platforms. Each MD region of αIIb, αL, and α5 was replaced with those of other α subunits that heterodimerize with β3, β2, and β1 integrins, respectively. β3 subunit forms heterodimers with αIIb and αV integrins. β2 subunit forms heterodimers with αL, αM, αD, and αX integrins. β1 subunit forms heterodimers with α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, and αV integrins. Thus, using these integrin α-chimeras, we were able to systemically study the role of 17 α-MD regions in integrin inside-out activation while retaining the native association of α and β subunits at the cytoplasmic domains. Ligand-mimetic mAb PAC-1, intercellular adhesion molecule-1 (ICAM-1), and human fibronectin were used to measure the talin-head-induced activation of αIIb, αL, and α5 chimeras co-expressed in HEK293FT cells with β3, β2, and β1 integrins, respectively. Conformation-specific monoclonal antibodies were used to report integrin conformational activation. The endogenous α5β1 integrin of HEK293FT cells were knocked out by the CRISPR/Cas9 technology. Our data showed that the chimeric α integrins had different levels of inside-out activation when compared with their corresponding wild-type integrins. Some chimeras such as αIIb-αV, αL-αX, αL-αD, αL-αM, α5-α2, α5-α4, and α5-α9 showed lower integrin activation than the wild types, while other chimeras such as α5-α7 and α5-α10 rendered α5β1 integrin more active than wild type. As a control, the αIIb-α1 and αIIb-αL chimeras all showed higher inside-out activation than wild-type αIIb. Our results suggest that specific amino acids of the α-MD region that immediately follow the GFFKR motif might contribute to integrin inside-out activation, probably through regulating the conformational change of the integrin α transmembrane and cytoplasmic domains. Our study demonstrates an important role of the α-MD region in integrin activation and indicates that structure and sequence diversity of the α-MD region might contribute to the diverse functions of integrins, which are determined by different integrin α subunits.
Disclosures
No relevant conflicts of interest to declare.
Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.