β1D integrin splice variant stabilizes integrin dynamics and reduces integrin signaling by limiting paxillin recruitment

Abstract

Heterodimeric integrin receptors control cell adhesion, migration and extracellular matrix assembly. While the α integrin subunit determines extracellular ligand specificity, the β integrin chain binds to an acidic residue of the ligand, and cytoplasmic adapter protein families such as talins, kindlins and paxillin, to form mechanosensing cell matrix adhesions. Alternative splicing of the β1 integrin cytoplasmic tail creates ubiquitously expressed β1A, and the heart and skeletal musclespecific β1D form. To study the physiological difference between these forms, we developed fluorescent β1 integrins and analyzed their dynamics, localization, and cytoplasmic adapter recruitment and effects on cell proliferation. On fibronectin, GFP-tagged β1A integrin showed dynamic exchange in peripheral focal adhesions, and long, central fibrillar adhesions. In contrast, GFP-β1D integrins exchanged slowly, forming immobile and short central adhesions. While adhesion recruitment of GFP-β1A integrin was sensitive to C-terminal tail mutagenesis, GFP-β1D integrin was recruited independently of the distal NPXY motif. In addition, a P786A mutation in the proximal, talin-binding NPXY 783 motif switched β1D to a highly dynamic integrin. In contrast, the inverse A786P mutation in β1A integrin interfered with paxillin recruitment and proliferation. Thus, differential β1 integrin splicing controls integrin-dependent adhesion signaling, to adapt to the specific physiological needs of differentiated muscle cells.

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to focal adhesions

“…Previous results supported an interaction of the paxillin LIM domains with the Y 747 sidechain, situated in the membrane-proximal NPLY 747 motif of β3 integrin 30,[41][42][43]48 . Although the dissociation rate of paxillin carrying an inactive LIM2 domain (paxillin 1334) was almost exactly that of paxillin wt dissociating from the β3 VE/YA integrin-containing adhesions (Fig.…”

Stimulation of Fibronectin Matrix Assembly by Lysine Acetylation

“…To test whether modification of K794 alters FN assembly, we generated two mutations at this site in the β1 integrin: lysine to glutamine (K794Q) to mimic an acetylated lysine and lysine to arginine (K794R) to prevent acetylation but retain a positively charged residue at that location [22]. In order to localize the transfected integrins, a GFP tag was also inserted into the extracellular domain of the integrin [33]. Wild type and mutant integrin constructs were transfected into a β1 integrin-null mouse fibroblast cell line (GD25) and stably transfected cells were isolated.…”

Induction of ligand promiscuity of αVβ3 integrin by mechanical force

“…At the same time, more than 12 potential RGD ligands for αVβ3 integrin have been reported (Humphries et al, 2006), but it remains unclear whether or how these ligands are selected by αVβ3 integrin. We have recently developed a method to produce microstructured Fn/vitronectin (Vn) substrates to analyze ligand selection by αVβ3 integrin on a cellular level (Pinon et al, 2014;Rahikainen et al, 2017;Soto-Ribeiro et al, 2019). Fn is a structural component of the ECM and has essential functions during development (George et al, 1993).…”