Low-affinity State Research Articles

Comparison of High and Low Affinity Serotonin 1A Receptors by PET In Vivo in Nonhuman Primates

Serotonin (5-HT) 1A receptors exist in high and low affinity states. Agonist ligands bind preferentially to the high affinity state receptors, providing a more functionally relevant measure than antagonist binding. We now report comparison of 5-HT1A binding in vivo using both [11C]CUMI-101 (agonist) and [11C]WAY100635 (antagonist) in nonhuman primates. PET studies show that both tracers bind to known 5-HT1A receptor (5-HT1AR)-rich regions of baboon brain. The binding (BPF) of [11C]CUMI-101 was lower on an average of 55% across the regions of interest (ROIs) compared to [11C]WAY100635. This ratio is consistent with the in vitro binding data of agonist and antagonist 5-HT1AR ligands previously reported.

How does Hemoglobin Create Such Diverse Functionality of Physiological Relevance?

Hemoglobin (Hb) without heterotropic effectors or stripped Hb, is an O2-carrier with a high-affinity (P50 100 mmHg, KR from 10 to 0.005 /mmHg, KT from 0.3 to 0.005 /mmHg, KR/KT from 1 to >500, and ΔH+ up to −4.4 H+/Hb. Such enhanced functionality of Hb of physiological relevance is accomplished through pH-dependent asymmetric binding of heterotropic effectors to R(oxy)- and T(deoxy)-quaternary structures of Hb, which cause pH-dependent differential modulations/reductions of KR and KT, respectively. No changes in high-resolution static crystallographic T(deoxy)- and R(oxy)-quaternary and tertiary structures of Hb and their heme environment, as well as the axial coordination structures of the deoxy-heme (νFe-His = 215 /cm) and the oxy-heme (νFe-O2 = 567/cm) in solution, are observed, despite KT and KR values are changed as much as 100- and 2,000-folds, respectively. Thus, the assumption that the low-affinity state is caused by the inter-dimeric salt-bridge-linked constraints, the out-of-plane shift of the heme Fe, and the allosteric core constraint in the T(deoxy)-Hb is no longer valid. Although these constraints are completely absent in R(oxy)-Hb, its O2-affinity is modulated as much as 2,000-folds by its interaction with heterotropic effectors. The effector-linked modulation of thermal fluctuation of the protein may be responsible.

Increased affinity of dopamine for D2‐like versus D1‐like receptors. Relevance for volume transmission in interpreting PET findings

Evidence indicates that dopamine (DA) mainly acts as a volume transmission (VT) transmitter through its release into the extracellular fluid where the D(1) -like and D(2) -like receptors are predominantly extrasynaptic. It was therefore of interest to compare the affinities of the two major families of DA receptors. [(3)H] raclopride /DA and [(3)H] SCH23390/DA competition assays compared the affinity of DA at D(2) -like and D(1) -like receptors in rat dorsal striatal membrane preparations as well as in membrane preparations from CHO cell lines stably transfected with human D(2L) and D(1) receptors. The IC(50) values of DA at D(2) -like receptors in dorsal striatal membranes and CHO cell membranes were markedly and significantly reduced compared with the IC(50) values of DA at D(1) -like receptors. These IC(50) values reflect differences in both the high and low affinity states. The K(iH) value for DA at [(3)H] raclopride-labeled D(2) -like receptors in dorsal striatum was 12 nM, and this can help explain PET findings that amphetamine-induced increases in DA release can produce an up to 50% decrease of [(11)C] raclopride binding in the dorsal striatum in vivo. These combined results give indications for the existence of striatal D(2) -like receptor-mediated DA VT at the local circuit level in vivo. The demonstration of a K(iH) value of 183 nM for DA at D(1) antagonist-labeled D(1) -like receptors instead gives a likely explanation for the failure of a reduction of D(1) -like receptor binding after amphetamine-induced DA release in PET studies using the D(1) -like antagonist radioligands [(11)C] SCH23390 and [(11)C] NNC. It seems difficult to evaluate the role of the extrasynaptic D(1) receptors in VT in vivo with the PET radioligands available for this receptor.

The PLATELET INTEGRIN αIIbβ3 CHANGES FROM A LOWER- to A Higher-AFFINITY STATE DURING INTERACTION with FIBRINOGEN

Abstract 1130The integrin αIIbβ3 (GPIIb-IIIa) plays an essential role in platelet adhesion and cohesion by binding to a number of adhesive ligands including fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Following ligand binding, αIIbβ3-mediated intracellular signaling is thought to stabilize platelet adhesive interactions, cause platelet spreading, and initiate clot retraction. Here we investigated the time course of interactions between purified αIIbβ3 and its major ligand fibrinogen when both were firmly attached to apposed surfaces, mimicking αIIbβ3-mediated platelet adhesive interactions. To measure the mechanical resistance of individual αIIbβ3-fibrinogen complexes under a constant tensile force, we used a previously described optical trap force clamp system (Litvinov et al., Biophys. J., 2011, 100, 165). Briefly, we trapped a fibrinogen-coated microscopic bead in a focused laser beam and intermittently brought it into contact with an αIIbβ3-coated silica pedestal. Repeated touching and separation of the bead and pedestal occurred with a compressive force of 20 pN and a constant pulling force of 50 pN. When the duration of contact between interacting molecules on the bead and pedestal was varied from 0.1s to 2s, the probability of αIIbβ3-fibrinogen interaction was time-dependent. This enabled us to extract two-dimensional kinetic parameters, i.e., how fast the αIIbβ3-fibrinogen complex forms and dissociates at the interface without the application of an external tensile force (zero-force kinetics). To convert the kinetic parameters to absolute values, we determined the density of αIIbβ3 molecules on the pedestal surface capable of binding 125I-fibrinogen to be ≈3,000 molecules per μm2. We found that the reactive αIIbβ3 exists in at least two states that differ significantly in their on-rates (kon1 =8×10−5 and kon2 =5.6×10−4 μm2/s), off-rates (koff1 =1.56 and koff2 =1.70 1/s) and affinities (Kd1 =2×104 and Kd2 =3×103 1/μm2) for fibrinogen. In the absence of αIIbβ3 activators, the interacting molecules reached a stable equilibrium in which 85% of the complexes were in the lower affinity form. In the presence of the activator Mn2+, the proportion of lower affinity forms decreased to 60%. Mn2+ also changed the on-rates (kon1 =3.38×10−5 and kon2 =9.9×10−4 μm2/s), off-rates (koff1 =2.99 and koff2 =4.82 1/s) and affinities (Kd1 =9×104 and Kd2 =5×103 1/μm2). We then tested whether the ratio between the two integrin activation states and the mechanical stability of the αIIbβ3-fibrinogen complex depends on the duration of contact between the interacting surfaces. As the contact duration was prolonged from 0.1s to 2s, the αIIbβ3-fibrinogen bond lifetime increased from shorter lifetimes (<2s) corresponding to the low-affinity state to longer lifetimes (up to 50s) corresponding to the high-affinity state. Moreover, the rate of transition from low- to high-affinity increased ∼3-fold, whereas the reverse rate decreased 1.5–2-fold, resulting in an overall 6-fold increase in the equilibrium rate constant. This means that reversible conformational transitions of αIIbβ3 from low- to high affinity states occur during its interaction with fibrinogen with the equilibrium gradually shifting toward the higher affinity state as contact duration increases. Thus, the strength of αIIbβ3-fibrinogen interactions is time-dependent due to a progressive increase in the affinity of the αIIbβ3-fibrinogen complex during the course of the interaction. These results provide kinetic and thermodynamic characteristics of the adhesive interaction between αIIbβ3 and fibrinogen at the single-molecule level and provide evidence for a direct fibrinogen-induced change in αIIbβ3 conformation as a potential mechanism for the initiation of αIIbβ3-mediated outside-in signaling in platelets. Disclosures:No relevant conflicts of interest to declare.

Imaging the high-affinity state of the dopamine D 2 receptor in vivo: Fact or fiction?

Positron Emission Tomography (PET) has been used for more than three decades to image and quantify dopamine D 2 receptors (D 2R) in vivo with antagonist radioligands but in the recent years agonist radioligands have also been employed. In vitro competition studies have demonstrated that agonists bind to both a high and a low-affinity state of the D 2Rs, of which the high affinity state reflects receptors that are coupled to G-proteins and the low-affinity state reflects receptors uncoupled from G-proteins. In contrast, antagonists bind with uniform affinity to the total pool of receptors. Results of these studies led to the proposal that D 2Rs exist in high and low-affinity states for agonists in vivo and sparked the development and use of agonist radioligands for PET imaging with the primary purpose of measuring the proportion of receptors in the high-affinity (activating) state. Although several lines of research support the presence of high and low-affinity states of D 2Rs and their detection by in vivo imaging paradigms, a growing body of controversial data has now called this into question. These include both in vivo and ex vivo studies of anesthesia effects, rodent models with increased proportions of high-affinity state D 2Rs as well as the molecular evidence for stable receptor–G-protein complexes. In this commentary we review these data and discuss the evidence for the in vivo existence of D 2Rs configured in high and low-affinity states and whether or not the high-affinity state of the D 2R can, in fact, be imaged in vivo with agonist radioligands.

Identification of Integrin β Subunit Mutations That Alter Affinity for Extracellular Matrix Ligand

We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5' end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNAiMet) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an "open" conformation of the 40S subunit with Met-tRNAiMet bound in a low-affinity state conducive to scanning and in triggering rearrangement into a "closed" conformation incompatible with scanning, which features Met-tRNAiMet more tightly bound to the "P" site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded "landing pad" for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.

Antagonists show GTP‐sensitive high‐affinity binding to the sigma‐1 receptor

Sigma-1 receptors are atypical receptors with potentially two transmembrane domains. Antagonists require doses significantly higher than their published affinities to have biological effects. We have reassessed the binding characteristics of these ligands and found antagonists bind to high- and low-affinity states not distinguished by agonists. The affinities of sigma-1 receptor ligands was assessed using radioligand saturation and competition binding of [³H]-(+)-pentazocine to permeabilized MDA-MB-468 cells. This was compared with the effect of ligands on metabolic activity using an MTS-based assay and calcium signalling using cells loaded with the calcium dye, Fura-2. Sigma-1 receptor antagonists, but not agonists, show GTP- and suramin-sensitive high-affinity binding. Functional responses (calcium signalling and metabolic activity), while associated with sigma-1 receptor binding, required binding to an unidentified, low-affinity target. Sigma-1 receptors are coupled to G proteins. This interaction is only observed when analysing antagonist binding. The identity of the G protein remains to be resolved. The concept of agonist and antagonist at the sigma-1 receptor needs to be revisited.

Robust Spindle Alignment in Drosophila Neuroblasts by Ultrasensitive Activation of Pins

Cellular signaling pathways exhibit complex response profiles with features such as thresholds and steep activation (i.e., ultrasensitivity). In a reconstituted mitotic spindle orientation pathway, activation of Drosophila Pins (LGN in mammals) by Gαi is ultrasensitive (apparent Hill coefficient of 3.1), such that Pins recruitment of the microtubule binding protein Mud (NuMA) occurs over a very narrow Gαi concentration range. Ultrasensitivity is required for Pins function in neuroblasts as a nonultrasensitive Pins mutant fails to robustly couple spindle position to cell polarity. Pins contains three Gαi binding GoLoco domains (GLs); Gαi binding to GL3 activates Pins, whereas GLs 1 and 2 shape the response profile. Although cooperative binding is one mechanism for generating ultrasensitivity, we find GLs 1 and 2 act as "decoys" that compete against activation at GL3. Many signaling proteins contain multiple protein interaction domains, and the decoy mechanism may be a common method for generating ultrasensitivity in regulatory pathways.

Type 1 Fimbrial Adhesin FimH Elicits an Immune Response That Enhances Cell Adhesion of Escherichia coli

Escherichia coli causes about 90% of urinary tract infections (UTI), and more than 95% of all UTI-causing E. coli express type 1 fimbriae. The fimbrial tip-positioned adhesive protein FimH utilizes a shear force-enhanced, so-called catch-bond mechanism of interaction with its receptor, mannose, where the lectin domain of FimH shifts from a low- to a high-affinity conformation upon separation from the anchoring pilin domain. Here, we show that immunization with the lectin domain induces antibodies that exclusively or predominantly recognize only the high-affinity conformation. In the lectin domain, we identified four high-affinity-specific epitopes, all positioned away from the mannose-binding pocket, which are recognized by 20 separate clones of monoclonal antibody. None of the monoclonal or polyclonal antibodies against the lectin domain inhibited the adhesive function. On the contrary, the antibodies enhanced FimH-mediated binding to mannosylated ligands and increased by severalfold bacterial adhesion to urothelial cells. Furthermore, by natural conversion from the high- to the low-affinity state, FimH adhesin was able to shed the antibodies bound to it. When whole fimbriae were used, the antifimbrial immune serum that contained a significant amount of antibodies against the lectin domain of FimH was also able to enhance FimH-mediated binding. Thus, bacterial adhesins (or other surface antigens) with the ability to switch between alternative conformations have the potential to induce a conformation-specific immune response that has a function-enhancing rather than -inhibiting impact on the protein. These observations have implications for the development of adhesin-specific vaccines and may serve as a paradigm for antibody-mediated enhancement of pathogen binding.

Synthesis, biophysical properties, and oxygenation potential of variable molecular weight glutaraldehyde‐polymerized bovine hemoglobins with low and high oxygen affinity

In a recent study, ultrahigh molecular weight (Mw ) glutaraldehyde-polymerized bovine hemoglobins (PolybHbs) were synthesized with low O2 affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top-load model.(1) In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G:Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O2 affinity state. Our results showed that the Mw of the resulting PolybHbs increased with increasing G:Hb molar ratio. For low O2 affinity PolybHbs, increasing the G:Hb molar ratio reduced the O2 affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O2 affinity PolybHbs, increasing the G:Hb molar ratio led to increased O2 affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O2 affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G:Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G:Hb molar ratio. To preliminarily evaluate the ability of low and high O2 affinity PolybHbs to potentially oxygenate tissues in vivo, an O2 transport model was used to simulate O2 transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O2 affinity PolybHbs oxygenated the bioreactor better than high O2 affinity PolybHbs. This result points to the suitability of low O2 affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G:Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin-based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications.

Regulation of Integrin αIIbβ3 Ligand Binding and Signaling by the Metal Ion Binding Sites in the β I Domain

The ability of αIIbβ3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the β I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the β I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the β3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain.

Allosteric Mechanism of Oxygen-Binding in Hemoglobin

The widely held structure-based mechanism of cooperative oxygen-binding in hemoglobin (Hb), in which the oxygen-affinity of Hb is regulated by the T to R-quaternary and associated tertiary structural transition, is based upon the assumption of the structure-function correlation: ([deoxy-state] =[T-quaternary structure]=[low-affinity state]) and ([oxy-state]=[R-quaternary structure]= [high-affinity state]. Simultaneous measurements of the quaternary structure by proton NMR and the oxygen-affinity by oxygen-binding equilibrium have yielded the following structural/functional states of Hb: (IV) T(deoxy)Hb with extreme low-affinity, (V) R(oxy)Hb with extreme low-affinity, and (VI) T(oxy)Hb with extreme low-affinity in the presence of potent heterotropic effectors as well as more conventional (II) T(deoxy)Hb with low-affinity and (III) R(oxy)Hb with high-affinity in the absence of heterotropic effectors. These results indicate the above-mentioned assumed structure-function correlation is no longer valid. The structure-based allosteric mechanism of Hb describes merely ligation-linked structural allostery rather than the cooperative mechanism of Hb. The structure and the function ( the oxygen-affinity, the cooperativty, and the Bohr effect) of Hb are regulated independently by a tug of war between the allosteric effects of oxygen (T-->R and increasing affinity) and the opposing allosteric effects of heterotropic effectors (T<--R and decreasing affinity) rather than the T to R-quaternary and associated tertiary structural transition. The oxygen-affinity is regulated by effector-linked dynamic structural changes.

The Molecular Chaperone Hsp70 Family Members Function by a Bidirectional Heterotrophic Allosteric Mechanism

The Hsp70 family is one of the most important and conserved molecular chaperone families. It is well documented that Hsp70 family members assist many cellular processes involving protein quality control, as follows: protein folding, transport through membranes, protein degradation, escape from aggregation, intracellular signaling, among several others. The Hsp70 proteins act as a cellular pivot capable of receiving and distributing substrates among the other molecular chaperone families. Despite the high identity of the Hsp70 proteins, there are several homologue Hsp70 members that do not have the same role in the cell, which allow them to develop and participate in such large number of activities. The Hsp70 proteins are composed of two main domains: one that binds ATP and hydrolyses it to ADP and another which directly interacts with substrates. These domains present bidirectional heterotrophic allosteric regulation allowing a fine regulated cycle of substrate binding and release. The general mechanism of the Hsp70s cycle is under the control of ATP hydrolysis that modulates the low (ATP-bound state) and high (ADP-bound state) affinity states of Hsp70 for substrates. An important feature of the Hsp70s cycle is that they have several co-chaperones that modulate their cycle and that can also interact and select substrates. Here, we review some known details of the bidirectional heterotrophic allosteric mechanism and other important features for Hsp70s regulating cycle and function.

Mechanism of Binding Site Conformational Switching in the CD44–Hyaluronan Protein–Carbohydrate Binding Interaction

The transmembrane protein CD44, which has been implicated in cancer biology and inflammation, mediates cell adhesion through multimeric interactions with the linear extracellular glycosaminoglycan hyaluronan (HA; in megadaltons). Affinity switching of CD44 from a low-affinity state to a high-affinity state is required for normal CD44 physiological function; crystal structures of the CD44 hyaluronan binding domain complexed with HA oligomers point to a conformational rearrangement at a binding site loop, leading to the formation of direct contact between the oligomer and an arginine side chain as a molecular basis for affinity switching. Here, all-atom explicit-solvent molecular dynamics simulations were used to characterize the dynamics and thermodynamics of oligomeric hyaluronan (oHA) and its two crystallographic complexes with the CD44 hyaluronan binding domain: the “A-form,” which lacks arginine–HA close contact, and the “B-form,” which has direct arginine side-chain–HA contact. From the simulations, the conformational properties of oHA are essentially unaltered in going from the unbound state to either the A-form or the B-form bound state, with the oligomer retaining its flexibility when bound and with only two of the eight monosaccharides in the oligomer maintaining uninterrupted contact with the protein. Biased simulations revealed that altering the backbone conformation of a tyrosine residue in the arginine loop can induce the A-form → B-form conformational transition and that a large free-energy barrier prevents ready interconversion between the two forms, thereby suggesting that the tyrosine backbone forms a molecular switch.

A potential role for α-actinin in inside-out αIIbβ3 signaling

AbstractMany different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbβ3, whereas inside-out signaling (αIIbβ3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbβ3. We evaluated the time-dependent changes of the αIIbβ3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbβ3 activation, whereas PAR4-activating peptide induced long-lasting αIIbβ3 activation. When αIIbβ3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbβ3. Compared with control platelets, the dissociation of α-actinin from αIIbβ3 was only transient in PAR4-stimulated P2Y12-deficient platelets in which the sustained αIIbβ3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbβ3 and inhibited PAR1-induced initial αIIbβ3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbβ3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbβ3 activation by inside-out signaling.

Sticky and Sweet: The Role of Post-Translational Modifications on Neisserial Pili

Sticky and Sweet: The Role of Post-Translational Modifications on Neisserial Pili

Activation State of α4β1 Integrin on Sickle Red Blood Cells Is Linked to the Duffy Antigen Receptor for Chemokines (DARC) Expression

In sickle cell anemia, reticulocytes express enhanced levels of α4β1 integrin that interact mainly with vascular cell adhesion molecule-1 and fibronectin, promoting vaso-occlusion. These interactions are known to be highly sensitive to the inflammatory chemokine IL-8. The Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes. However, the link between α4β1 activation by chemokines and DARC erythroid expression is not or poorly explored. Therefore, the capacity of α4β1 to mediate Duffy-negative and Duffy-positive sickle reticulocyte (SRe) adhesion to immobilized vascular cell adhesion molecule-1 and fibronectin was evaluated. Using static adhesion assays, we found that, under basal conditions, Duffy-positive SRe adhesion was 2-fold higher than that of Duffy-negative SRes. Incubating the cells with IL-8 or RANTES (regulated on activation normal T cell expressed and secreted) increased Duffy-positive SRe adhesion only, whereas Mn(2+) increased cell adhesion independently of the Duffy phenotype. Flow cytometry analyses performed with anti-β1 and anti-α4 antibodies, including a conformation-sensitive one, in the presence or absence of IL-8, revealed that Duffy-positive and Duffy-negative SRes displayed similar erythroid α4β1 expression levels, but with distinct activation states. IL-8 did not affect α4β1 affinity in Duffy-positive SRes but induced its clustering as corroborated by immunofluorescence microscopy. Our results indicate that in Duffy-negative SRes α4β1 integrin is constitutively expressed in a low affinity state, whereas in Duffy-positive SRes α4β1 is expressed in a higher chemokine-sensitive affinity state. This activation state associated with DARC RBC expression may influence the intensity of the inflammatory responses encountered in sickle cell anemia and participate in its interindividual clinical expression variability.

Identification of Interacting Hot Spots in the β3 Integrin Stalk Using Comprehensive Interface Design

Protein-protein interfaces are usually large and complementary surfaces, but specific side chains, representing energetic "hot spots," often contribute disproportionately to binding free energy. We used a computational method, comprehensive interface design, to identify hot spots in the interface between the stalk regions of the β3 and the complementary αIIb and αv integrin subunits. Using the Rosetta alanine-scanning and design algorithms to predict destabilizing, stabilizing, and neutral mutations in the β3 region extending from residues Lys(532) through Gly(690), we predicted eight alanine mutations that would destabilize the αIIbβ3 interface as well as nine predicted to destabilize the αvβ3 interface, by at least 0.3 kcal/mol. The mutations were widely and unevenly distributed, with four between residues 552 and 563 and five between 590 and 610, but none between 565 and 589, and 611 and 655. Further, mutations destabilizing the αvβ3 and αIIbβ3 interfaces were not identical. The predictions were then tested by introducing selected mutations into the full-length integrins expressed in Chinese hamster ovary cells. Five mutations predicted to destabilize αIIb and β3 caused fibrinogen binding to αIIbβ3, whereas three of four predicted to be neutral or stabilizing did not. Conversely, a mutation predicted to destabilize αvβ3, but not αIIbβ3 (D552A), caused osteopontin binding to αvβ3, but not fibrinogen binding to αIIbβ3. These results indicate that stability of the distal stalk interface is involved in constraining integrins in stable, inactive conformations. Further, they demonstrate the ability of comprehensive interface design to identify functionally significant integrin mutations.

Millimolar Mn2+ influences agonist binding to 5-HT1A receptors by inhibiting guanosine nucleotide binding to receptor-coupled G-proteins

Manganese is an essential trace element but its overexposure causes poisoning (called manganism) that shares several symptoms with Parkinson's disease, but with a mechanism that is still not well understood: in addition to involvement of the dopaminergic system, both serotonergic and peptiergic systems have been implicated. In the present report we have studied the influence of Mn2+ on 5-HT1A receptor signaling complexes in rat brain and found that Mn2+ in millimolar concentration caused an increase of high-affinity agonist binding to rat hippocampal membranes in comparison with experiments in the presence of Mg2+, but not in rat cortical membranes and in Sf9 cell membranes expressing 5-HT1A receptors and Gi1 heterotrimers. Activation of G proteins with 30μM GTPγS turned all 5-HT1A receptors in these preparations into a low-affinity state for agonist binding in the presence of 1mM Mg2+, but not in the presence of 1mM Mn2+ in rat hippocampal membranes. However, if 1μM GTPγS was used for G protein activation, a substantial amount of high affinity agonist binding was detected in the presence of Mn2+ also in cortical membranes and Sf9 cells, but not with Mg2+ or EDTA. Comparison of the abilities of GDP and GTPγS to modulate high affinity agonist binding to 5-HT1A receptors indicated that both nucleotides were almost 10-fold less potent in the presence of MnCl2 compared to MgCl2. This means that by inhibiting guanosine nucleotide binding to G proteins in complex with 5-HT1A receptors, Mn2+ acts as an enhancer for agonist binding and signal transduction. As the influence of Mn2+ resembles the hypersensitivity of dopaminergic system in Parkinsonial models, it can be proposed that at least some symptoms of manganism are connected with a change of signal transduction complex caused by manganese-nucleotide complexes.