A 17-residue Sequence from the Matrix Metalloproteinase-9 (MMP-9) Hemopexin Domain Binds α4β1 Integrin and Inhibits MMP-9-induced Functions in Chronic Lymphocytic Leukemia B Cells

We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4β1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4β1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC(50) values of 138 and 279 μm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesis.     

Structural Determinants of G-protein �� Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2)

“Regulator of G-protein signaling” (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Gα subunits (known as GTPase-accelerating protein or “GAP” activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Gα_q subunits. As many vasoconstrictive hormones signal via G_q heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Gα_q over Gα_i/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Gα_i-directed binding and GAP activities without perturbing its association with Gα_q. Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Gα_i at 2.8-Å resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Gα complexes highlighted the roles of these residues in wild type RGS2 that weaken Gα_i subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Gα_q GAP that modulates cardiovascular function.G protein-coupled receptors (GPCRs)⁴ form an interface between extracellular and intracellular physiology, as they convert hormonal signals into changes in intracellular metabolism and ultimately cell phenotype and function (1–3). GPCRs are coupled to their underlying second messenger systems by heterotrimeric guanine nucleotide-binding protein (“G-proteins”) composed of three subunits: Gα, Gβ, and Gγ. Four general classes of Gα subunits have been defined based on functional couplings (in the GTP-bound state) to various effector proteins. G_s subfamily Gα subunits are stimulatory to membrane-bound adenylyl cyclases that generate the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP); conversely, G_i subfamily Gα subunits are generally inhibitory to adenylyl cyclases (4). G_12/13 subfamily Gα subunits activate the small G-protein RhoA through stimulation of the GEF subfamily of RGS proteins, namely p115-RhoGEF, LARG, and PDZ-RhoGEF (5). G_q subfamily Gα subunits are potent activators of phospholipase-Cβ enzymes that generate the second messengers diacylglycerol and inositol triphosphate (6); more recently, two additional Gα_q effector proteins have been described: the receptor kinase GRK2 and the RhoA nucleotide exchange factor p63RhoGEF (7, 8).The duration of GPCR signaling is controlled by the time Gα remains bound to GTP before its hydrolysis to GDP. RGS proteins are key modulators of GPCR signaling by virtue of their ability to accelerate the intrinsic GTP hydrolysis activity of Gα subunits (reviewed in Refs. 9 and 10). RGS2/G0S8, one of the first mammalian RGS proteins identified (11) and member of the R4-subfamily (10), has a critical role in the maintenance of normostatic blood pressure both in mouse models (12, 13) and in humans (14, 15); additionally, Rgs2-deficient mice exhibit impaired aggression and increased anxiety (16, 17), behavioral phenotypes with potential human clinical correlates (18, 19).Although many RGS proteins are promiscuous and thus act on multiple Gα substrates in vitro (e.g. Ref. 20), RGS2 exhibits exquisite specificity for Gα_q in biochemical binding assays and single turnover GTPase acceleration assays (20, 21). Consistent with this in vitro selectivity,⁵ mice deficient in RGS2 uniquely exhibit constitutive hypertension and prolonged responses to vasoconstrictors, as would be expected upon loss of a potent negative regulator of Gα_q that mediates signaling from various vasoconstrictive hormones such as angiotensin II, endothelin, thrombin, norepinephrine, and vasopressin (22). In addition, RGS2-deficient mice respond to sustained pressure overload with an accelerated time course of maladaptive cardiac remodeling (23), a pathophysiological response that evokes myocardial hypertrophy known to be critically dependent on Gα_q signaling (24, 25).To gain insight into the structural basis of the unique Gα substrate selectivity exhibited by RGS2, a series of point mutants in RGS2 were evaluated that enable this protein to bind and accelerate GTP hydrolysis by Gα_i; we subsequently delineated the structural determinants of the Gα_i/mutant RGS2 interaction using x-ray crystallography. Three key positions, first identified by Heximer and colleagues (21) and highlighted in our structural studies as key determinants of RGS2 substrate selection, were also found to be conserved throughout the evolution of the RGS2 protein in a manner suggestive of specialization toward cardiovascular signaling modulation.     

Reversible Inactivation of Purified Leukocyte Integrin CR3 (CD11b/CD18, βmβ2) by Removal of Divalent Cations from a Cryptic Site

Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, α_mβ₂) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that “inactive” CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca²⁺ and Mg²⁺, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg²⁺ with an affinity (17.8 ± 4.5 nM) similar to untreated, active receptor (12.5 ± 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg²⁺-binding region in the α chain of CR3. We found that monomeric C3bi binds directly to recombinant I-domain in a Mg²⁺-dependent fashion with an affinity of 300 ± 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3.     

The Dermatan Sulfate Proteoglycan Decorin Modulates ��2��1 Integrin and the Vimentin Intermediate Filament System during Collagen Synthesis

Decorin, a small leucine-rich proteoglycan harboring a dermatan sulfate chain at its N-terminus, is involved in regulating matrix organization and cell signaling. Loss of the dermatan sulfate of decorin leads to an Ehlers-Danlos syndrome characterized by delayed wound healing. Decorin-null (Dcn^−/−) mice display a phenotype similar to that of EDS patients. The fibrillar collagen phenotype of Dcn^−/− mice could be rescued in vitro by decorin but not with decorin lacking the glycosaminoglycan chain. We utilized a 3D cell culture model to investigate the impact of the altered extracellular matrix on Dcn^−/− fibroblasts. Using 2D gel electrophoresis followed by mass spectrometry, we identified vimentin as one of the proteins that was differentially upregulated by the presence of decorin. We discovered that a decorin-deficient matrix leads to abnormal nuclear morphology in the Dcn^−/− fibroblasts. This phenotype could be rescued by the decorin proteoglycan but less efficiently by the decorin protein core. Decorin treatment led to a significant reduction of the α2β1 integrin at day 6 in Dcn^−/− fibroblasts, whereas the protein core had no effect on β1. Interestingly, only the decorin core induced mRNA synthesis, phosphorylation and de novo synthesis of vimentin indicating that the proteoglycan decorin in the extracellular matrix stabilizes the vimentin intermediate filament system. We could support these results in vivo, because the dermis of wild-type mice have more vimentin and less β1 integrin compared to Dcn^−/−. Furthermore, the α2β1 null fibroblasts also showed a reduced amount of vimentin compared to wild-type. These data show for the first time that decorin has an impact on the biology of α2β1 integrin and the vimentin intermediate filament system. Moreover, our findings provide a mechanistic explanation for the reported defects in wound healing associated with the Dcn^−/− phenotype.     

The leucocyte β2 (CD18) integrins: the structure, functional regulation and signalling properties

Leucocytes are highly motile cells. Their ability to migrate into tissues and organs is dependent on cell adhesion molecules. The integrins are a family of heterodimeric transmembrane cell adhesion molecules that are also signalling receptors. They are involved in many biological processes, including the development of metazoans, immunity, haemostasis, wound healing and cell survival, proliferation and differentiation. The leucocyte-restricted β2 integrins comprise four members, namely αLβ2, αMβ2, αXβ2 and αDβ2, which are required for a functional immune system. In this paper, the structure, functional regulation and signalling properties of these integrins are reviewed.     

Audiogram of the chicken (Gallus gallus domesticus) from 2 Hz to 9 kHz

The pure-tone thresholds of four domestic female chickens were determined from 2 Hz to 9 kHz using the method of conditioned suppression/avoidance. At a level of 60 dB sound pressure level (re 20 μN/m²), their hearing range extends from 9.1 Hz to 7.2 kHz, with a best sensitivity of 2.6 dB at 2 kHz. Chickens have better sensitivity than humans for frequencies below 64 Hz; indeed, their sensitivity to infrasound exceeds that of the homing pigeon. However, when threshold testing moved to the lower frequencies, the animals required additional training before their final thresholds were obtained, suggesting that they may perceive frequencies below 64 Hz differently than higher frequencies.     

Extracellular Matrix Lumican Deposited on the Surface of Neutrophils Promotes Migration by Binding to ��2 Integrin

During inflammation, circulating polymorphonuclear neutrophils (PMNs) receive signals to cross the endothelial barrier and migrate through the extracellular matrix (ECM) to reach the injured site. Migration requires complex and poorly understood interactions of chemokines, chemokine receptors, ECM molecules, integrins, and other receptors. Here we show that the ECM protein lumican regulates PMN migration through interactions with specific integrin receptors. Lumican-deficient (Lum^−/−) mice manifest connective tissue defects, impaired innate immune response, and poor wound healing with reduced PMN infiltration. Lum^−/− PMNs exhibit poor chemotactic migration that is restored with exogenous recombinant lumican and inhibited by anti-lumican antibody, confirming a role for lumican in PMN migration. Treatment of PMNs with antibodies that block β₂, β₁, and α_M integrin subunits inhibits lumican-mediated migration. Furthermore, immunohistochemical and biochemical approaches indicate binding of lumican to β₂, α_M, and α_L integrin subunits. Thus, lumican may regulate PMN migration mediated by MAC-1 (α_M/β₂) and LFA-1 (α_L/β₂), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.Polymorphonuclear neutrophils (PMNs)³ play a major role in the development of inflammatory responses to host injury and infection. Their functions include destruction of invading bacteria and recruitment of macrophages and lymphocytes to the affected site (1). Circulating PMNs sense injury and pathogen signals, cross the vascular endothelium, and migrate to the target tissue; two series of events control this process. The first leads to the slowing down and adherence of circulating PMNs on the vascular endothelium followed by their transendothelial migration or extravasation and activation (2). The second controls the directional migration of PMNs to the injured site through the endothelial basement membrane, a specialized type of ECM, and subsequently the deeper interstitial ECM, along chemokine and cytokine gradients. Leukocyte-to-leukocyte and leukocyte-to-endothelium interactions are important before extravasation. These are mediated by interactions between selectins and their ligands and by β₂ (MAC-1 and LFA-1) and β₁ (VLA-4–6) integrin interactions with cell adhesion proteins ICAM and PECAM (3). The directional migration of PMNs through the ECM is a complex, multistep process that involves several α and β integrin interactions with ECM proteins. Thus far, a few basement membrane proteins, laminins, entactin, and fibronectin have been identified as specific ligands in regulating migration of PMNs after extravasation (4–6). Additional interstitial ECM proteins and their receptors that modulate PMN migration have yet to be identified. Here we show that the ECM protein lumican is a novel regulator of PMN migration.Lumican is a secreted collagen-binding ECM protein of the corneal, dermal, and tendon stroma, arterial wall, and the intestinal submucosa (7–9). It is a member of the small leucine-rich repeat proteoglycans (10); these were initially investigated in the context of binding collagen and regulating tissue structure and biomechanics (11, 12). A body of literature is beginning to indicate that these proteoglycans interact with cytokines, growth factors, and cell surface receptors to modulate cell adhesion, proliferation, and migration (13–16). Lumican and biglycan, another member of this family of proteoglycans, have been recently shown to regulate host response to pathogen-associated molecular patterns (17, 18). Thus, lumican-deficient (Lum^−/−) mice are hyporesponsive to bacterial lipopolysaccharide (LPS) endotoxins, and Lum^−/− macrophages in culture produce lower levels of pro-inflammatory cytokines in response to LPS (18). Lumican facilitates innate immune response by binding LPS and CD14, the glycerol phosphatidylinositol-linked cell surface adaptor protein that transfers the LPS signal to toll-like receptor 4 (18). In a corneal injury model neutrophil influx is delayed in the Lum^−/− mice (19, 20). Although this may be partly due to impaired innate immune response, it raises the possibility that lumican may have an additional role in neutrophil migration. Here we elucidate a role for lumican in PMN migration. We show that poor chemotactic migration of Lum^−/− PMNs can be rescued by exogenous recombinant lumican (rLum) and blocked specifically with antibodies against lumican, β₂, β₁, and α_M integrins. Our results also show that lumican localizes on the surface of extravasated PMNs through its interactions with β₂ integrins. The likely source of lumican on neutrophils is the vascular endothelium.