Low-affinity State Research Articles

Thermodynamics of oxygenation-linked proton and lactate binding govern the temperature sensitivity of O2 binding in crustacean(Carcinus maenas) hemocyanin

With the aim of understanding the molecular underpinnings of the enormous variation in the temperature sensitivity of hemocyanin-O2 affinity encountered in crustaceans, we measured O2 binding to Carcinus maenas hemocyanin at two temperatures, varying pH values and in the absence and presence of lactate ions in order to assess the contributions of oxygenation-linked binding of protons (the Bohr effect) and lactate ions to the overall enthalpies of oxygenation (DeltaH'). The hemocyanin binds maximally 0.35 lactate ions per functional subunit. Lactate (which accumulates under hypoxic conditions) increases O2 affinity by preferentially raising the association equilibrium constant of the hemocyanin in the low-affinity Tense state (KT), without significantly affecting that of the high-affinity Relaxed state (KR). In the absence of lactate, the variation in the temperature sensitivity observed with decreasing pH tallies neatly with changes in the nature and magnitude of the Bohr effect. Accordingly, the normal, absent and reverse Bohr effects observed under alkaline, neutral and acid conditions, respectively, reflect endothermic proton dissociation, absence of proton binding and exothermic proton association, respectively, upon oxygen binding. Oxygenation-linked lactate binding is exothermic, highly pH dependent and peaks near pH 7.6, where it contributes approximately -30 kJ mol(-1) to the overall heat of oxygenation. This predictably increases the temperature sensitivity of O2 affinity, potentially hampering O2 loading in warm, hypoxic habitats. The data demonstrate governing roles for lactate and proton ions in determining the temperature sensitivity of hemocyanin-O2 affinity in crustaceans.

Molecular mechanisms that control initiation and termination of physiological depolarization-evoked transmitter release

Ca(2+) is essential for physiological depolarization-evoked synchronous neurotransmitter release. But, whether Ca(2+) influx or another factor controls release initiation is still under debate. The time course of ACh release is controlled by a presynaptic inhibitory G protein-coupled autoreceptor (GPCR), whose agonist-binding affinity is voltage-sensitive. However, the relevance of this property for release control is not known. To resolve this question, we used pertussis toxin (PTX), which uncouples GPCR from its G(i/o) and in turn reduces the affinity of GPCR toward its agonist. We show that PTX enhances ACh and glutamate release (in mice and crayfish, respectively) and, most importantly, alters the time course of release without affecting Ca(2+) currents. These effects are not mediated by G(beta)gamma because its microinjection into the presynaptic terminal did not alter the time course of release. Also, PTX reduces the association of the GPCR with the exocytotic machinery, and this association is restored by the addition of agonist. We offer the following mechanism for control of initiation and termination of physiological depolarization-evoked transmitter release. At rest, release is under tonic block achieved by the transmitter-bound high-affinity presynaptic GPCR interacting with the exocytotic machinery. Upon depolarization, the GPCR uncouples from its G protein and consequently shifts to a low-affinity state toward the transmitter. The transmitter dissociates, the unbound GPCR detaches from the exocytotic machinery, and the tonic block is alleviated. The free machinery, together with Ca(2+) that had already entered, initiates release. Release terminates when the reverse occurs upon repolarization.

An alternative theoretical formula for hemoglobin oxygenation

Classical models of homotropic allostery are based on the postulate that the binding sites are equivalent in their ability to interconvert between high and low affinity states, but compelling evidence exists that the subunits of human hemoglobin are not simultaneously available for oxygen equilibration, thus reducing the number of possible intermediate microstates. The incorporation of these results into the Adair scheme reveals an alternative mechanism for hemoglobin oxygenation, not based on affinity changes.

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.

Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo

Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin β subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.

Adenosine A1 receptor‐mediated inhibition of dopamine release from rat striatal slices is modulated by D1 dopamine receptors

Dopamine release is regulated by presynaptic dopamine receptors and interactions between adenosine and dopamine receptors have been well documented. In the present study, dopamine release from isolated striatal slices from Wistar rats was measured using fast cyclic voltammetry. Single-pulse stimulation (0.1 ms, 10 V) was applied every 5 min over a 2-h period. Superfusion with the adenosine (A)(1) receptor agonist N(6)-cyclopentyladenosine (CPA), but not the A(2) receptor agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl] phenyl]propanoic acid (CGS 21680), inhibited dopamine release in a concentration-dependent manner (IC(50) 3.80 x 10(-7) m; n = 10). The dose-response curve to CPA was shifted to the right (IC(50) 6.57 x 10(-6) m; n = 6, P < 0.05 vs. control) by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Neither the D(1) agonist 6-chloro-APB nor the D(1) antagonist R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3- benzazepine-7-ol (SCH 23390) altered dopamine release on their own. However, SCH 23390 (3 microm) significantly attenuated the response to CPA (IC(50) 1.44 x 10(-5) m; n = 6, P < 0.01 vs. control). Furthermore, the inhibitory effect of CPA was significantly increased in the presence of 6-chloro-APB (1 microm). In radioligand binding experiments, CPA interacted with high- and low-affinity states of [(3)H]DPCPX-lableled A(1) receptors. The high-affinity agonist binding to A(1) receptors was inhibited by the stable guanosine triphosphate analogue Gpp(NH)p. In contrast, neither the proportion nor the affinity of high-affinity A(1) receptors was altered by dopamine or SCH 23390. These results provide evidence that the inhibition of dopamine release by adenosine A(1) receptors is dependent, at least in part, on the simultaneous activation of D(1) dopamine receptors. While the mechanism underlying this interaction remains to be determined, it does not appear to involve an intramembrane interaction between A(1) and D(1) receptors.

Characterization of G-Protein Coupled Receptor Kinase Interaction with the Neurokinin-1 Receptor Using Bioluminescence Resonance Energy Transfer

To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET(2) saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.

Single-Molecule Analysis of Nucleotide-Dependent Substrate Binding by the Protein Unfoldase ClpA

Single-molecule fluorescence experiments on the ClpA ATPase component of the ClpAP protease demonstrate that ClpA binds its peptide substrates in discrete high- and low-affinity conformations. These conformations correspond to different states in the catalytic cycle of ATP hydrolysis. On the basis of these observations, we propose that translocation of substrates through the central pore of ClpA is driven by a switch between high- and low-affinity states.

Antiparkinson therapeutic potencies correlate with their affinities at dopamine D2High receptors

To determine whether antiparkinson dopamine agonists preferentially act on the high-affinity or the low-affinity states of dopamine D1 and D2 receptors, the agonist potencies were obtained by competition against [(3)H]SCH23390 for D1(High) and D1(Low), and against [(3)H]domperidone for D2(High) and D2(Low). N-propylnorapomorphine and cabergoline were the most potent at D2(High), with dissociation constants of 0.18 and 0.36 nM, respectively. Other agonists had D2(High)K(i) values of 0.52 nM for quinagolide, 0.6 nM for (+)PHNO, 0.9 for bromocriptine, 1.8 nM for apomorphine, 2.4 nM for pergolide, 3 nM for quinpirole, and 6.2 nM for lergotrile. There was a clear correlation between the K(i) values at D2(High) and their therapeutic concentrations in the plasma water, as derived from the known concentrations after correction for the fraction bound to the human plasma proteins. The data suggest that D2(High) is the primary and common target for the antiparkinson action of dopamine agonists. Bromocriptine, cabergoline, lergotrile, pergolide, and pramipexole had no affinity for D1(High), consistent with the clinical observations that the D2-selective bromocriptine and pramipexole elicit low levels of dyskinesia.

A Structural Hypothesis for the Transition between Bent and Extended Conformations of the Leukocyte β2 Integrins

Integrins mediate cell adhesion in response to activation signals that trigger conformational changes within their ectodomain. It is thought that a compact bent conformation of the molecule represents its physiological low affinity state and extended conformations its active state. We have determined the structure of two integrin fragments of the beta2 subunit. The first structure, consisting of the plexin-semaphorin-integrin domain, hybrid, integrin-epidermal growth factor 1 (I-EGF1), and I-EGF2 domains (PHE2), showed an L-shaped conformation with the bend located between the I-EGF1 and I-EGF2 domains. The second structure, which includes, in addition, the I-EGF3 domain, showed an extended conformation. The major reorientation of I-EGF2 with respect to the other domains in the two structures is accompanied by a change of torsion angle of the disulfide bond between Cys(461)-Cys(492) by 180 degrees and the conversion of a short alpha-helix (residues Ser(468)-Cys(475)) into a flexible coil. Based on the PHE2 structure, we introduced a disulfide bond between the plexin-semaphorin-integrin domain and I-EGF2 domains in the beta2 subunit. The resultant alphaLbeta2 integrin (leukocyte function-associated antigen-1) variant was locked in a bent state and could not be detected with the monoclonal antibody KIM127 in Mg(2+)/EGTA. However, it retained the binding activity to ICAM-1. These results provide a structural hypothesis for our understanding of the transition between the resting and active states of leukocyte function-associated antigen-1.

Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m(7)Guo), and orthophosphate (P(i), natural cosubstrate) and its thiophosphate analogue (SP(i)), found to be a weak cosubstrate, have been studied in the pH range 5-8. In this pH range Guo and Ino exist predominantly in the neutral forms (pK(a) 9.2 and 8.8); m(7)Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pK(a) 7.0); and P(i) and SP(i) exhibit equilibria between monoanionic and dianionic forms (pK(a) 6.7 and 5.4, respectively). The phosphorolysis of m(7)Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SP(i), independently of pH. With P(i), the human enzyme shows Michaelis kinetics only at pH approximately 5. However, in the pH range 5-8 for the bacterial enzyme, and 6-8 for the human enzyme, enzyme kinetics with P(i) are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by P(i), characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K (m1) and K (m2)) and apparent maximal velocities (V (max1) and V (max2)). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K (m1) < K (m2)) and enzyme kinetics (V (max1)/K (m1) > V (max2)/K (m2)). Comparison of the pH-dependence of the substrate properties of P(i) versus SP(i) points to both monoanionic and dianionic forms of P(i) as substrates, with a marked preference for the dianionic species in the pH range 5-8, where the population of the P(i) dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n (H) approximately 1 to n (H) approximately 0.7 for Guo with the human enzyme, and to n (H) approximately 0.7 and 0.5 for m(7)Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SP(i) in relation to its structure.

Comparative Normal Mode Analysis of LFA-1 Integrin I-domains

The conformational dynamics of the Inserted domain (I-domain) from the lymphocyte function-associated antigen-1 (LFA-1) was investigated by normal mode analysis of multiple structures of the low, intermediate, and high affinity states. LFA-1 is an integrin expressed on leukocytes and is of critical importance in adhesion reactions, like antigen-specific responses, homing, and diapedesis. The main ligand binding site of LFA-1 is the I-domain, which recognizes intercellular adhesion molecules (ICAMs), members of the immunoglobulin superfamily. From experimental crystal structures, a large-scale conformational change of, among others, the α7 helix of the I-domain has been observed leading to the proposal that these structural changes are linked to the conformational regulation of LFA-1. The results from the present calculations show that structural changes of the α7 helix consistent with those observed in the crystal structures are significantly sampled by the low frequency modes. This was found to be particularly true for the low affinity state of the I-domain, indicating that low frequency motions favor the conformational transition implicated in activation. However, beyond the simple downward shift of the helix implied by the crystal structures, the calculations further show that there is a noticeable swinging-out motion of the helix. The consequences of this motion are discussed in the context of integrin activation and inhibition. Moreover, significant changes in the atomic-level dynamics and in long-range correlated motions of the I-domain were found to occur upon binding of the natural ligand ICAM. These changes were more local upon binding of an allosteric inhibitor. The present study opens the question of how changes in dynamics may contribute to the long-range transmission of signal upon ICAM binding by the LFA-1 I-domain.

Brain region binding of the D2/3 agonist [11C]‐(+)‐PHNO and the D2/3 antagonist [11C]raclopride in healthy humans

The D(2) receptors exist in either the high- or low-affinity state with respect to agonists, and while agonists bind preferentially to the high-affinity state, antagonists do not distinguish between the two states. [(11)C]-(+)-PHNO is a PET D(2) agonist radioligand and therefore provides a preferential measure of the D(2) (high) receptors. In contrast, [(11)C]raclopride is an antagonist radioligand and thus binds with equal affinity to the D(2) high- and low-affinity states. The aim was to compare the brain uptake, distribution and binding characteristics between [(11)C]-(+)-PHNO and [(11)C]raclopride in volunteers using a within-subject design. Both radioligands accumulated in brain areas rich in D(2)/D(3)-receptors. However, [(11)C]-(+)-PHNO showed preferential uptake in the ventral striatum and globus pallidus, while [(11)C]raclopride showed preferential uptake in the dorsal striatum. Mean binding potentials were higher in the putamen (4.3 vs. 2.8) and caudate (3.4 vs 2.1) for [(11)C]raclopride, equal in the ventral-striatum (3.4 vs. 3.3), and higher in the globus pallidus for [(11)C]-(+)-PHNO (1.8 vs. 3.3). Moreover [(11)C]-(+)-PHNO kinetics in the globus pallidus showed a slower washout than other regions. One explanation for the preferential binding of [(11)C]-(+)-PHNO in the globus pallidus and ventral-striatum could be the presence of a greater proportion of high- vs. low-affinity receptors in these areas. Alternatively, the observed distribution could also be explained by a preferential binding of D(3)-over-D(2) with [(11)C]-(+)-PHNO. This differential binding of agonist vs. antagonist radioligand, especially in the critically important region of the limbic striatum/pallidum, offers new avenues to investigate the role of the dopamine system in health and disease.

Mapping early conformational changes in αIIb and β3 during biogenesis reveals a potential mechanism for αIIbβ3 adopting its bent conformation

AbstractCurrent evidence supports a model in which the low-affinity state of the platelet integrin αIIbβ3 results from αIIbβ3 adopting a bent conformation. To assess αIIbβ3 biogenesis and how αIIbβ3 initially adopts the bent conformation, we mapped the conformational states occupied by αIIb and β3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that αIIbβ3 complex formation was not limited by the availability of either free pro-αIIb or free β3, suggesting that other molecules, perhaps chaperones, control complex formation. Five β3-specific, ligand-induced binding site (LIBS) mAbs reacted with much or all free β3 but not with β3 when in complex with mature αIIb, suggesting that β3 adopts its mature conformation only after complex formation. Conversely, 2 αIIb-specific LIBS mAbs directed against the αIIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-αIIb, raising the possibility that pro-αIIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-αIIb adopts a bent conformation soon after synthesis, and then β3 assumes its bent conformation by virtue of its interaction with the bent pro-αIIb.

First Human Evidence of d-Amphetamine Induced Displacement of a D2/3 Agonist Radioligand: A [11C]-(+)-PHNO Positron Emission Tomography Study

Imaging the competition between D(2/3) radioligands and endogenous dopamine is so far the only way to measure dopamine release in the living human brain. The dopamine D(2) receptor exists in a high (D(2)(high)) and a low-affinity state for dopamine. Under physiological conditions, dopamine is expected to bind to D(2)(high) only. [(11)C]-(+)-4-propyl-9-hydroxynaphthoxazine ((+)-PHNO) is the first D(2/3) agonist radioligand for positron emission tomography (PET) imaging in humans. Since [(11)C]-(+)-PHNO is expected to bind preferentially to D(2)(high), it should be particularly vulnerable to competition with endogenous dopamine. Nine healthy subjects participated in two PET scans, one after administration of d-amphetamine and one after placebo. [(11)C]-(+)-PHNO PET test re-test variability was determined in 11 healthy subjects. Binding potentials (BPs) were calculated for caudate, putamen, ventral striatum, and globus pallidus. d-Amphetamine led to a significant decrease of [(11)C]-(+)-PHNO BPs in caudate (-13.2%), putamen (-20.8%), and ventral striatum (-24.9%), but not in globus pallidus (-6.5%). d-Amphetamine-induced displacement correlated with serum d-amphetamine levels in all regions but caudate. This is the first report on competition between endogenous dopamine and a D(2/3) agonist radioligand in humans. [(11)C]-(+)-PHNO PET might be a superior measure for release of endogenous dopamine than PET employing conventional D(2/3) antagonist radioligands.

Tests of the Extension and Deadbolt Models of Integrin Activation

Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the beta3 tail domain could act as a deadbolt to restrain the displacement of the beta3 I domain beta6-alpha7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the beta3 tail domain loop has no effect on ligand binding by either alphaIIbbeta 3 or alphaVbeta3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation.

Slow conformational dynamics of the guanine nucleotide‐binding protein Ras complexed with the GTP analogue GTPγS

The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) leads to (31)P NMR spectra that indicate the prevalence of only one conformational state with K(12) > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTPgammaS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy DeltaH(|) (1a1b) of 61 kJ.mol(-1) and an activation entropy DeltaS(|) (1a1b) of 65 J.K(-1).mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K(A) for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTPgammaS complex.

Protean Agonism at the Dopamine D2 Receptor: (S)-3-(3-Hydroxyphenyl)-N-propylpiperidine Is an Agonist for Activation of Go1 but an Antagonist/Inverse Agonist for Gi1,Gi2, and Gi3

A range of ligands displayed agonism at the long isoform of the human dopamine D(2) receptor, whether using receptor-G protein fusions or membranes of cells in which pertussis toxin-resistant mutants of individual Galpha(i)-family G proteins could be expressed in an inducible fashion. Varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [(35)S]GTPgammaS onto each of Galpha(i1),Galpha(i2),Galpha(i3), and Galpha(o1). By contrast, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine was a partial agonist when Galpha(o1) was the target G protein but an antagonist/inverse agonist at Galpha(i1),Galpha(i2), and Galpha(i3). In ligand binding assays, dopamine identified both high- and low-affinity states at each of the dopamine D(2) receptor-G protein fusion proteins, and the high-affinity state was eliminated by guanine nucleotide. (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine bound to an apparent single state of the constructs in which the D(2) receptor was fused to Galpha(i1),Galpha(i2), or Galpha(i3). However, it bound to distinct high- and low-affinity states of the D(2) receptor-Galpha(o1) fusion, with the high-affinity state being eliminated by guanine nucleotide. Likewise, although dopamine identified guanine nucleotide-sensitive high-affinity states of the D(2) receptor when expression of pertussis toxin-resistant forms of each of Galpha(i1), Galpha(i2), Galpha(i3), and Galpha(o1) was induced, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine identified a high-affinity site only in the presence of Galpha(o1). p-Tyramine displayed a protean ligand profile similar to that of (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine but with lower potency. These results demonstrate (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine to be a protean agonist at the D(2) receptor and may explain in vivo actions of this ligand.

309: Blocking LFA-1 activation with lovastatin prevents graft-versus-host disease in mouse bone marrow transplantation

Leukocyte function associated antigen-1 (LFA-1) regulates T cell adhesion and activation. LFA-1 is constitutively expressed on cell surface in an inactive state. The control of LFA-1 activation is critical in inflammatory and immune responses. We demonstrated previously that the I-domain, the ligand binding site of LFA-1, changes from the low-affinity state to high-affinity state upon activation. Therapeutic antagonist, such as lovastatin, stabilizes the I-domain in the low-affinity state and inhibits the LFA-1 activation. Here, we report that lovastatin can block mouse T cell adhesion and proliferation in vitro. First, we demonstrated that lovastatin treatment reduced the mortality and morbidity in the mouse GVHD model. Lovastatin treatment significantly decreased GVHD mortality with 80% mice survived over 28 days, whereas more than 70% of the control mice died within the first 10 days, and the p values was 0.045. There were significantly reduced tissue damages in the skin, intestine and liver of lovastatin-treated mice. Second, we found lovastatin treatment reduced donor T cell homing to lymph nodes. There was a 65% reduction of CD4+ T cells homing to lymph nodes in lovastatin treatment group compared to control. The reduction of CD8+ T cells was greater with about 76% less cells homing to lymph nodes in the lovastatin treatment group. Third, we found lovastatin treatment reduced donor-derived T cell proliferation in vivo. There were 37% CD4+ and 31% CD8+ T cells remained undivided in the lymph nodes of the control mice at day 4 post-transplant. The lovastatin-treated mice had reduced proliferation kinetics of both CD4+ and CD8+ T cells with about 55% and 42% remained undivided. In the control lymph nodes, there were 42% CD4+ and 59% CD8+ T cells proliferated beyond 5th and 6th cell-divisions respectively, whereas the lovastatin treatment reduced the number to 31% and 48% respectively. In summary, we demonstrated here that lovastatin prevents both homing and proliferating of donor-derived T cells in the secondary lymphoid organs, which are crucial sites for alloreactive expansion. While most of the control mice died of acute GVHD within the first week of post-transplant when alloreactive T cells infiltrated the targeted organs, lovastatin treatment prevented the activation and expansion of donor-derived T cells, and thus reduced the GVHD mortality and morbidity. Our study provides rationale for a potential novel treatment for GVHD.

Syntheses and in vitro evaluation of fluorinated naphthoxazines as dopamine D2/D3 receptor agonists: radiosynthesis, ex vivo biodistribution and autoradiography of [18F]F-PHNO

Carbon-11-labeled (+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ([(11)C]-(+)-PHNO) is a dopamine D2/D3 agonist radioligand that is currently used to image the high-affinity state of dopamine receptors in humans with positron emission tomography (PET). The present study reports the preparation and evaluation of fluorinated (+)-PHNO derivatives. Five fluorinated (+)-PHNO derivatives were synthesized and tested in vitro for inhibition of binding of [(3)H]domperidone in homogenates of rat striatum and inhibition of binding to [(3)H]-(+)-PHNO in homogenates of human-cloned D2Long receptors in Chinese hamster ovary cells and rat striatum. Radiolabeling with fluorine-18 was carried out for the most promising candidate, N-fluoropropyl-(+)-HNO (F-PHNO), and ex vivo biodistribution and autoradiography studies with this radiopharmaceutical were performed in rodents. (+)-PHNO and the fluorinated analogs inhibited binding of [(3)H]domperidone and [(3)H]-(+)-PHNO to the high- and low-affinity states of dopamine D2 receptors, consistent with D2 agonist behavior. The average dissociation constant at the high-affinity state of D2, K(i)(High), was 0.4 nM for F-PHNO and proved to be equipotent with (+)-PHNO (0.7 nM). All other fluorinated derivatives were significantly less potent (K(i)(High)=2-102 nM). The most promising candidate, F-PHNO, was labeled with fluorine-18 in 5% uncorrected radiochemical yield, with respect to starting fluoride. Ex vivo biodistribution and autoradiography studies in rodents revealed that [(18)F]F-PHNO rapidly enters the rodent brain. However, this radiotracer does not reveal specific binding in the brain and is rapidly cleared. Five novel dopamine D2/D3 agonists based on (+)-PHNO were synthesized and evaluated in vitro. F-PHNO was shown to behave as a potent D2 agonist in vitro and was therefore radiolabeled with fluorine-18. Despite the promising in vitro pharmacological profile, [(18)F]F-PHNO did not display in vivo behavior suitable to image dopaminergic receptor expression using PET.