Regulation Of Integrin Activation Research Articles

Abstract 4380: Role of kindlin-2 in prostate cancer progression and metastasis

Abstract Prostate cancer (PC) is the leading type of cancer in men accounting for more than 25% of all newly reported cancer cases and remains the second leading cause of cancer-related deaths in men in USA in 2023. The success of early treatments has resulted in a 98% 5-year survival rate for early-stage PC. However, resistance to androgen ablation can lead to metastatic castration-resistant prostate cancer (mCRPC), with a significant drop in the survival rate to ~30%. The metastatic cascade involves changes in adhesive, metabolic and signaling properties of PC cells, allowing them to survive, proliferate and thrive in their new microenvironment. Kindlin-2 (K2), a cytoskeletal adapter protein, is highly expressed in cancer, including PC cells and tumors, contributing to their tumorigenic properties. The present study focuses on three K2 functions relevant to PC metastasis: 1)Reduction in K2 levels, achieved through CRISPR/Cas9, profoundly affected the tumorigenic properties of PC cell lines LNCaP (androgen dependent), DU145, and PC3 (both androgen independent), influencing colony formation, invasion, and adhesion to integrin ligands. While reduction in K2 was observed, K1 and K3 levels in these cell lines was not altered by K2KO. 2)The E3 ubiquitin ligase, Parkin, known for its role in Parkinson’s disease, has emerged as a tumor suppressor inhibiting various cancer hallmarks. Our recently published studies showed that Parkin induces ubiquitination of specific lysines in K2, leading to the hypothesis that Parkin acts as a tumor suppressor by targeting K2 for degradation. Stable over-expression of Parkin inhibited adhesion to fibrinogen (Fg), while loss of function Parkin mutants failed to do so, supporting a role of Parkin in K2 turnover and its impact on mitochondrial dynamics. We are currently investigating the effect of K2-lysines mutants as well as loss of function Parkin mutants on the progression and metastasis of PC tumors in vivo.3)K2's established function is the regulation of integrin activation, affecting adhesion receptors' transition from low to high affinity/avidity states. The K2-integrin axis is pivotal for understanding oncogenic responses, and its role in the prostate microenvironment in PC progression and metastasis is under exploration using our newly established K2-floxed mouse. The K2-floxed mice are currently being crossed with mice harboring prostate-epithelial specific cre under control of the probasin (Pbsn) promoter. These animal studies will shed light on K2's contribution to PC development and progression in vivo, providing insights into its role in the prostate microenvironment. To sum up, this study emphasizes the crucial role of K2 in the tumorigenic properties of PC cells and tumors. By incorporating CRISPR/Cas9 and innovative mouse models, the study seeks to shed the lights on the mechanisms through which K2 contributes to PC development and progression. Citation Format: Lamyae El Khalki, Wei Wang, Neelum A. Yousaf Zai, Justin Szpendyk, Elzbieta Pluskota, Katarzyna Bialkowska, Edward F. Plow, Lucia R. Languino, Dario C. Altieri, Khalid Sossey-Alaoui. Role of kindlin-2 in prostate cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4380.

PB0399 The NMDA Receptor Regulates Integrin Activation and Arterial Thrombosis through Store-Operated Ca2+ Entry in Platelets

PB0399 The NMDA Receptor Regulates Integrin Activation and Arterial Thrombosis through Store-Operated Ca2+ Entry in Platelets

LRP12 is an endogenous transmembrane inactivator of α4 integrins

Dynamic regulation of integrin activation and inactivation is critical for precisely controlled cell adhesion and migration in physiological and pathological processes. The molecular basis for integrin activation has been intensively studied; however, the understanding of integrin inactivation is still limited. Here, we identify LRP12 as an endogenous transmembrane inhibitor for α4 integrin activation. The LRP12 cytoplasmic domain directly binds to the integrin α4 cytoplasmic tail and inhibits talin binding to the β subunit, thus keeping integrin inactive. In migrating cells, LRP12-α4 interaction induces nascent adhesion (NA) turnover at the leading-edge protrusion. Knockdown of LRP12 leads to increased NAs and enhanced cell migration. Consistently, LRP12-deficient Tcells show an enhanced homing capability in mice and lead to aggravated chronic colitis in a Tcell-transfer colitis model. Altogether, LRP12 is a transmembrane inactivator for integrins that inhibits α4 integrin activation and controls cell migration by maintaining balanced NA dynamics.

Multi-functional adaptor SKAP1: regulator of integrin activation, the stop-signal, and the proliferation of T cells.

T-cell activation is a complex process involving a network of kinases and downstream molecular scaffolds or adaptors that integrate surface signals with effector functions. One key immune-specific adaptor is Src kinase-associated phosphoprotein 1 (SKAP1), which is also known as src kinase-associated protein of 55 kDa (SKAP55). This mini-review explains how SKAP1 plays multiple roles in regulating integrin activation, the "stop-signal", and the optimization of the cell cycling of proliferating T cells through interactions with various mediators, including the Polo-like kinase 1 (PLK1). Ongoing research on SKAP1 and its binding partners will likely provide important insights into the regulation of immune function and have implications for the development of new treatments for disease states such as cancer and autoimmunity.

The NMDA receptor regulates integrin activation, ATP release and arterial thrombosis through store-operated Ca2+ entry in platelets.

Platelet activation and thrombus formation is crucial for hemostasis, but also trigger arterial thrombosis. Calcium mobilization plays an important role in platelet activation, because many cellular processes depend on the level of intracellular Ca2+ ([Ca2+](i)), such as integrin activation, degranulation, cytoskeletal reorganization. Different modulators of Ca2+ signaling have been implied, such as STIM1, Orai1, CyPA, SGK1, etc. Also, the N-methyl-D-aspartate receptor (NMDAR) was identified to contribute to Ca2+ signaling in platelets. However, the role of the NMDAR in thrombus formation is not well defined. In vitro and in vivo analysis of platelet-specific NMDAR knock-out mice. In this study, we analyzed Grin1fl/fl-Pf4-Cre+ mice with a platelet-specific knock-out of the essential GluN1 subunit of the NMDAR. We found reduced store-operated Ca2+ entry (SOCE), but unaltered store release in GluN1-deficient platelets. Defective SOCE resulted in reduced Src and PKC substrate phosphorylation following stimulation of glycoprotein (GP)VI or the thrombin receptor PAR4 followed by decreased integrin activation but unaltered degranulation. Consequently, thrombus formation on collagen under flow conditions was reduced ex vivo, and Grin1fl/fl-Pf4-Cre+ mice were protected against arterial thrombosis. Results from human platelets treated with the NMDAR antagonist MK-801 revealed a crucial role of the NMDAR in integrin activation and Ca2+ homeostasis in human platelets as well. NMDAR signaling is important for SOCE in platelets and contributes to platelet activation and arterial thrombosis. Thus, the NMDAR represents a novel target for anti-platelet therapy in cardiovascular disease (CVD).

Integrins in GtoPdb v.2023.1

Integrins are unusual signalling proteins that function to signal both from the extracellular environment into the cell, but also from the cytoplasm to the external of the cell. The intracellular signalling cascades associated with integrin activation focus on protein kinase activities, such as focal adhesion kinase and Src. Based on this association between extracellular signals and intracellular protein kinase activity, we have chosen to include integrins in the 'Catalytic receptors' section of the database until more stringent criteria from NC-IUPHAR allows precise definition of their classification.Integrins are heterodimeric entities, composed of α and β subunits, each 1TM proteins, which bind components of the extracellular matrix or counter-receptors expressed on other cells. One class of integrin contains an inserted domain (I) in its α subunit, and if present (in α1, α2, α10, α11, αD, αE, αL, αM and αX), this I domain contains the ligand binding site. All β subunits possess a similar I-like domain, which has the capacity to bind ligand, often recognising the RGD motif. The presence of an α subunit I domain precludes ligand binding through the β subunit. Integrins provide a link between ligand and the actin cytoskeleton (through typically short intracellular domains). Integrins bind several divalent cations, including a Mg2+ ion in the I or I-like domain that is essential for ligand binding. Other cation binding sites may regulate integrin activity or stabilise the 3D structure. Integrins regulate the activity of particular protein kinases, including focal adhesion kinase and integrin-linked kinase. Cellular activation regulates integrin ligand affinity via inside-out signalling and ligand binding to integrins can regulate cellular activity via outside-in signalling.Several drugs that target integrins are in clinical use including: (1) abciximab (αIIbβ3) for short term prevention of coronary thrombosis, (2) vedolizumab (α4β7) to reduce gastrointestinal inflammation, and (3) natalizumab (α4β1) in some cases of severe multiple sclerosis.

A Novel Deletion in FERMT3 Causes LAD-III in a Turkish Family.

Leukocyte adhesion deficiency-III (LAD-III) is an extremely rare autosomal recessive syndrome caused by mutations in FERMT3, the gene encoding kindlin-3. The genetic alterations in this gene lead to abnormal expression or activity of kindlin-3 in leukocytes and platelets. Kindlin-3 acts as an important regulator of integrin activation. LAD-III has features of the bleeding syndrome of Glanzmann and also of leukocyte adhesion deficiency. In this study, we report on two families, one of Turkish and one of Syrian origin, with clinical features of LAD-III, loss of kindlin-3 protein expression, and a functional leukocyte defect. A novel, homozygous deletion in FERMT3 (c.921delC, p.Ser307Argfs*21) was found in the Turkish patient. The parents were carriers of the mutation, consistent with an autosomal recessive inheritance. A common c.1525C > T (p.Arg509*) mutation was found in the Syrian patient. In conclusion, beside the variant c.1525C > T in the FERMT3 gene, which was previously found in more than 15 patients in Anatolia, our study is the first to identify the novel homozygous variant c.921delC in the FERMT3 gene.

β1 integrin monoclonal antibody treatment ameliorates cerebral cavernous malformations.

β1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active β1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of β1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting β1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.

KRIT1-mediated regulation of neutrophil adhesion and motility.

Loss of Krev interaction-trapped-1 (KRIT1) expression leads to the development of cerebral cavernous malformations (CCM), a disease in which abnormal blood vessel formation compromises the structure and function of the blood-brain barrier. The role of KRIT1 in regulating endothelial function is well-established. However, several studies have suggested that KRIT1 could also play a role in regulating nonendothelial cell types and, in particular, immune cells. In this study, we generated a mouse model with neutrophil-specific deletion of KRIT1 in order to investigate the effect of KRIT1 deficiency on neutrophil function. Neutrophils isolated from adult Ly6Gtm2621(cre)Arte Krit1flox/flox mice had a reduced ability to attach and spread on the extracellular matrix protein fibronectin and exhibited a subsequent increase in migration. However, adhesion to and migration on ICAM-1 was unchanged. In addition, we used a monomeric, fluorescently-labelled fragment of fibronectin to show that integrin activation is reduced in the absence of KRIT1 expression, though β1 integrin expression appears unchanged. Finally, neutrophil migration in response to lipopolysaccharide-induced inflammation in the lung was decreased, as shown by reduced cell number and myeloperoxidase activity in lavage samples from Krit1PMNKO mice. Altogether, we show that KRIT1 regulates neutrophil adhesion and migration, likely through regulation of integrin activation, which can lead to altered inflammatory responses in vivo.

Selectin-Mediated Signaling-Shedding Light on the Regulation of Integrin Activity in Neutrophils.

As a consequence of tissue injury or infection, neutrophils are recruited in a stepwise recruitment process from the bloodstream into the surrounding tissue. Selectins are a family of adhesion molecules comprised of L-, E-, and P-selectin. Differences in expression patterns, protein structure, and ligand binding characteristics mediate distinct functions of each selectin. Interactions of selectins and their counter-receptors mediate the first contact of neutrophils with the endothelium, as well as subsequent neutrophil rolling along the endothelial surface. For efficient neutrophil recruitment, activation of β2-integrins on the cell surface is essential. Integrin activation can be elicited via selectin- as well as chemokine-mediated inside-out signaling resulting in integrin conformational changes and clustering. Dysregulation of selectin-induced integrin activation on neutrophils is involved in the development of severe pathological disease conditions including leukocyte adhesion deficiency (LAD) syndromes in humans. Here, we review molecular mechanisms involved in selectin-mediated signaling pathways in neutrophils and their impact on integrin activation, neutrophil recruitment, and inflammatory diseases.

Kindlin1 As a Gender and Location-Specific Diagnostic Marker in Gastric Cancer Patients.

Background & Objective:Gastric cancer (GC) is considered one of the main reasons for cancer-related mortalities among Iranians. Kindlin-1 is an adhesion protein member of integrin-interacting proteins, regulating integrin activation through direct interaction with β-integrin. Therefore, kindlin-1 can be involved in the regulation of cell proliferation and adhesion. In the present study, we assessed the possible role of kindlin-1 in GC progression and metastasis. Methods: KINDLIN1 messenger RNA (mRNA) expression was assessed in tumor tissues from 80 GC patients in comparison with normal tissues using real-time polymerase chain reaction (PCR).Results:The levels of KINDLIN1 expressions were significantly correlated with sex (P=0.05) and tumor location (P=0.002). KINDLIN1 expression was also significantly associated with lymph node metastasis among the Helicobacter Pylori (HP)-negative cases (P=0.001). Moreover, a significant association between age and KINDLIN1 expression was observed among the HP-positive cases (P=0.039). Conclusion:In the present study, we introduced KINDLIN1 as a location-specific marker for cardia gastric carcinoma. Moreover, it was observed that KINDLIN1 could be used as a gender-dependent diagnostic marker of GC patients.

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

MiRNA-200c-3p targets talin-1 to regulate integrin-mediated cell adhesion

The ability of integrins on the cell surface to mediate cell adhesion to the extracellular matrix ligands is regulated by intracellular signaling cascades. During this signaling process, the talin (TLN) recruited to integrin cytoplasmic tails plays the critical role of the major adaptor protein to trigger integrin activation. Thus, intracellular levels of TLN are thought to determine integrin-mediated cellular functions. However, the epigenetic regulation of TLN expression and consequent modulation of integrin activation remain to be elucidated. Bioinformatics analysis led us to consider miR-200c-3p as a TLN1-targeting miRNA. To test this, we have generated miR-200c-3p-overexpressing and miR-200c-3p-underexpressing cell lines, including HEK293T, HCT116, and LNCaP cells. Overexpression of miR-200c-3p resulted in a remarkable decrease in the expression of TLN1, which was associated with the suppression of integrin-mediated cell adhesion to fibronectin. In contrast, the reduction in endogenous miR-200c-3p levels led to increased expression of TLN1 and enhanced cell adhesion to fibronectin and focal adhesion plaques formation. Moreover, miR-200c-3p was found to target TLN1 by binding to its 3′-untranslated region (UTR). Taken together, our data indicate that miR-200c-3p contributes to the regulation of integrin activation and cell adhesion via the targeting of TLN1.

Microrna-15a-5p Regulates Multiple Components of GPVI-Mediated Platelet and Megakaryocyte Activation Pathway

Introduction: Platelet activation by collagen is a major effector ofnormal hemostasis. Mutations in GPVI, the major collagen signaling receptor, cause platelet-type bleeding. Two single nucleotide polymorphisms in GPVI have been identified as risk factors for myocardial infarction. Elevated levels of GPVI have been reported in patients with coronary syndromes. GPVI and its signaling pathway components for platelet activation have become potential antithrombotic targets. Reports have indicated microRNAs (miRs) regulate platelet activation, but none have considered the mechanisms involving miR-mRNA associations regulating the GPVI-mediated platelet activation pathway.Aims: To identify and characterize miRs regulating GPVI-mediated megakaryocyte (MK) integrin activation.Methods : Candidate miRs were identified via a platelet function association study. miR function was assessed by knockdown or overexpression studies in MKs derived from umbilical cord blood (CB) CD34+ stem cells. MK αIIbβ3 activation was measured by flow cytometric quantification of PAC-1 binding after collagen-related peptide (CRP) stimulation. miR and mRNA quantification were performed using quantitative polymerase chain reaction.Results: Our P latelet R NA A nd e X pression (PRAX1) study identified 20 miRs associated with CRP-induced platelet aggregation (p<0.008; n=154). To minimize false positives, we considered only miRs expressed in both platelets and cultured MKs, reducing the potential candidates to 10. We focused on miR-15a-5p because it is negatively correlated with platelet activation through CRP (p=0.0075) (Fig 1A) and because miR-15a-5p targets BCL2, which is known to enhance CRP-induced platelet activation. miR-15a-5p was not found to be associated with platelet aggregation in response to arachidonic acid, ADP, PAR1-AP or PAR4-AP, suggesting specificity of miR-15a-5p for GPVI-mediated integrin activation pathway. Knock down of miR-15a-5p using locked nucleic acid (LNA) in CBMKs caused a ~38% increase in CRP-induced integrin-activation (p=0.0035; n=7) compared to the scrambled (Scr) LNA control (Fig 1B), presumably by releasing miR-15a-5p repression of activation. Fig 1B shows lentiviral overexpression of miR-15a-5p decreased CRP-induced integrin-activation by ~33% (p=0.0154; n=4) as compared to empty vector (EV) control. To understand how miR-15a-5p regulates integrin activation, we bioinformatically identified putative mRNA targets FYN, SRGN, FCER1G, MYLK and PRKCQ, predicted to be regulated by miR-15a- 5p and known as signaling molecules in the GPVI activation pathway. Of note, GPVI was not predicted to be a target of miR-15a-5p and BCL2 was not included because it has been studied before. Importantly, these target genes are positively associated with CRP-induced platelet aggregation (all p<0.004; n=154). The transcript levels of FYN, FCER1G, MYLK and PRKCQ were derepressed upon miR-15a-5p knockdown (Fig 1C) as compared to Scr LNA (all p<0.05; n>4). In miR-15a-5p overexpressed CBMKs, transcript levels of FYN, FCER1G and PRKCQ were decreased compared to EV (Fig 1D) (all p<0.05; n=4). SRGN only showed trends upon miR-15a-5p manipulation. MYB and ACTIN were used as positive and negative controls, respectively. Initial studies of miR-15a-5p knockdown showed a trend for increased FYN, FCER1G and PRKCQ protein levels (Fig 1E; n=3). miR activity requires binding to Argonaute2 (Ago2), which catalyzes translational repression and mRNA degradation. We demonstrated that Ago2 immunoprecipitation enriches miR-15a-5p and its target FYN, SRGN, FCER1G, MYLK and PRKCQ mRNAs (Fig 1F). These data suggest miR-15a-5p and its targets are processed through the Ago complex and thereby, are potential miR-mRNA pairs together regulating GPVI-mediated integrin activation pathway.Conclusions:miR-15a-5p negatively regulates GPVI-mediated MK integrin activation. miR-15a-5p appears to act as a ‘master-miR’ regulating multiple components of GPVI-mediated platelet and MK activation. As miRs are increasingly becoming clinical therapeutic targets, miR-15a-5p overexpression shows potential for treating thrombosis, whereas its inhibition could be effective in bleeding disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Structure of an Extended β Integrin

Integrins are a family of 24 α/β heterodimeric adhesion receptors that transmit bidirectional signals across the cell membrane. Each integrin subunit contains a large extracellular domain composed of headpiece and leg piece, a transmembrane domain and usually a short cytoplasmic tail. The α headpiece consists of the common β-propeller and thigh domains, and an extra αI domain inserted into the β-propeller domain in a subset of α subunits such as αL. The α leg piece includes calf-1 and -2 domains. The β headpiece contains βI, which is a structural homolog of αI domain, hybrid, PSI, and I-EGF1 domains. The β leg piece contains I-EGF2-4 and β-tail domains. Structural studies in the last 15 years, largely based on the two β3 integrins, αIIbβ3 and αVβ3, greatly advanced our understanding of integrin conformational regulation upon activation. The platelet specific αIIbβ3 plays an essential role in hemostasis and thrombosis, while the αVβ3 is important for the growth and survival of many cell types. In the resting state, integrin adopts a bent conformation with the headpiece folded onto the leg piece. Crystallographic and EM studies of αIIbβ3 and αVβ3 revealed two major conformational rearrangements, the swing-out motion of the hybrid domain, resulting in headpiece opening, and the switchblade-like movement of the headpiece from the leg domains, leading to integrin extension and leg separation. Despite the visualization of β3 extension by EM at very low resolution, a high-resolution extended β3 structure has been missing.A Leu33Pro polymorphism at the β3 PSI domain defines the human platelet alloantigen 1 (HPA-1) system. The mismatched HPA-1a/b (Leu33/Pro33) is a major cause of two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP) due to the generation of anti-HPA-1 alloantibodies. In addition, it has been controversial whether the Pro33 variant can enhance β3 activity, being a risk factor for thrombosis. It is also unknown whether the Leu33Pro substitution affects the conformation of PSI domain, which undergoes outward movement along with the headpiece opening and extension.It has been difficult to obtain the crystal structure of a full-length extended integrin largely due to the conformational heterogeneity and flexibility, especially in the lower leg domains. In this study, we engineered a chimeric β3 fragment, denoted as cβ3-E2, including the domains of PSI, hybrid, I-EGF1, I-EGF2, and the βI replaced with the αL αI domain, which enables the expression of β3 in the absence of α subunit. We determined the crystal structures of cβ3-E2 in both Leu33 and Pro33 forms. Strikingly, two conformations of cβ3-E2, an intermediate state and a fully extended state are simultaneously present in one crystal. The β3 extension occurs at the I-EGF1 and I-EGF2 interface, denoted as β-knee. Our structure revealed the structural changes of the flexible loop, C473-C486 or C1-C2 loop of I-EGF2 in the β-knee, which has been shown to modulate integrin activation by mutagenesis studies. No significant structural differences were observed between the PSI domains having Leu33 and Pro33. We next compared the ligand binding of β3-Leu33 and β3-Pro33 induced by Mn2+ or talin head domain. No significant differences were observed either in αIIbβ3 or αVβ3, indicating that the Leu33Pro substitution may not affect β3 integrin activation. However, the PSI C26-C38 loop harboring the Leu33Pro polymorphism moves closer to the I-EGF1 domain in the extended conformation. In addition, we found that a completely conserved arginine (Arg93 in β3) at the PSI/hybrid domain interface is important for maintaining the resting integrin state since the alanine or glutamine substitution renders β1, β2 and β3 integrin hyperactive. Interestingly, a naturally occurring β3-Arg93Gln substitution has been shown to disrupt the HPA-1a epitope. Finally, based on our cβ3-E2 structure, we built the extended full-length β3 structures with either closed or open headpiece, which can perfectly fit into the EM structures of extended αIIbβ3 with either closed or open headpiece, respectively. In summary, our study provides structural details of an extended β3 integrin, showing the conformational changes at the PSI, I-EGF1 and I-EGF2 domains, demonstrating their importance in regulating integrin activation and alloimmune response. DisclosuresNo relevant conflicts of interest to declare.

Functional Integrin Regulation Through Interactions with Tetraspanin CD9.

Integrins are adhesion receptors that mediate many intercellular and cell-extracellular matrix interactions with relevance in physiology and pathology. Unlike other cellular receptors, integrins critically require activation for ligand binding. Through interaction in cis with other molecules and the formation of tetraspanin-enriched membrane microdomains (TEMs), the tetraspanin CD9 regulates integrin activity and avidity. Here we present three techniques used to study CD9-integrin interactions and integrin activation.

The Integrin Adaptor Kindlin-3 Is Important for Development and Retention of Marginal Zone B Cells

The Integrin Adaptor Kindlin-3 Is Important for Development and Retention of Marginal Zone B Cells

LIM domain proteins Pinch1/2 regulate chondrogenesis and bone mass in mice

The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion. Here, we report that deleting Pinch1 in limb mesenchymal stem cells (MSCs) and Pinch2 globally (double knockout; dKO) in mice causes severe chondrodysplasia, while single mutant mice do not display marked defects. Pinch deletion decreases chondrocyte proliferation, accelerates cell differentiation and disrupts column formation. Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone (PZ) chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone (HZ) chondrocytes. Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes, reduces bone formation, and increases bone resorption, leading to low bone mass. In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes. Through its C-terminal region, Pinch1 interacts with Smad2/3 proteins. Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells (BMSCs). Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs. Interestingly, compared to those from single mutant mice, BMSCs from dKO mice express dramatically lower protein levels of β-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity. Finally, ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening. Collectively, our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.

Roles of Membrane Domains in Integrin-Mediated Cell Adhesion.

The composition and organization of the plasma membrane play important functional and regulatory roles in integrin signaling, which direct many physiological and pathological processes, such as development, wound healing, immunity, thrombosis, and cancer metastasis. Membranes are comprised of regions that are thick or thin owing to spontaneous partitioning of long-chain saturated lipids from short-chain polyunsaturated lipids into domains defined as ordered and liquid-disorder domains, respectively. Liquid-ordered domains are typically 100 nm in diameter and sometimes referred to as lipid rafts. We posit that integrin β senses membrane thickness and that mechanical force on the membrane regulates integrin activation through membrane thinning. This review examines what we know about the nature and mechanism of the interaction of integrins with the plasma membrane and its effects on regulating integrins and its binding partners.

Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state.

Kindlin-1, -2, and -3 directly bind integrin β cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin β cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.