AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.

RNA binding characteristics and overall topology of the vaccinia virus polyadenylation complex

mRNA 3′ polyadenylation is central to mRNA biogenesis in prokaryotes and eukaryotes, and is implicated in numerous aspects of mRNA metabolism, including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. However, due to the great complexity of the eukaryotic polyadenylation apparatus, the mechanisms of RNA 3 ′ end processing have remained elusive. Although the RNA processing reactions leading to polyadenylated messenger RNA have been studied in many systems, and much progress has been made, a complete understanding of the biochemistry of the poly(A) polymerase enzyme is still lacking. My research uses Vaccinia virus as a model system to gain a better understanding of this complicated polyadenylation process, which consist of RNA binding, catalysis and polymerase translocation. ^ Vaccinia virus replicates in the cytoplasm of its host cell, so it must employ its own poly(A) polymerase (PAP), a heterodimer of two virus encoded proteins, VP55 and VP39. VP55 is the catalytic subunit, adding 30 adenylates to a non-polyadenylated RNA in a rapid processive manner before abruptly changing to a slow, non-processive mode of adenylate addition and dissociating from the RNA. VP39 is the stimulatory subunit. It has no polyadenylation catalytic activity by itself, but when associated with VP55 it facilitates the semi-processive synthesis of tails several hundred adenylates in length. ^ Oligonucleotide selection and competition studies have shown that the heterodimer binds a minimal motif of (rU)2 (N)25 U, the “heterodimer binding motif”, within an oligonucleotide, and its primer selection for polyadenylation is base-type specific. ^ Crosslinking studies using photosensitive uridylate analogs show that within a VP55-VP39-primer ternary complex, VP55 comes into contact with all three required uridylates, while VP39 only contacts the downstream uridylate. Further studies, using a backbone-anchored photosensitive crosslinker show that both PAP subunits are in close proximity to the downstream −10 to −21 region of 50mer model primers containing the heterodimer binding motif. This equal crosslinking to both subunits suggests that the dimerization of VP55 and VP39 creates either a cleft or a channel between the two subunits through which this region of RNA passes. ^ Peptide mapping studies of VP39 covalently crosslinked to the oligonucleotide have identified residue R107 as the amino acid in close proximity to the −10 uridylate. This helps us project a conceptual model onto the known physical surface of this subunit. In the absence of any tertiary structural data for VP55, we have used a series of oligonucleotide selection assays, as well as crosslinking, nucleotide transfer assays, and gel shift assays to gain insight into the requirements for binding, polyadenylation and translocation. Collectively, these data allow us to put together a comprehensive model of the structure and function of the polyadenylation ternary complex consisting of VP39, VP55 and RNA. ^

Role of transducer proteins in photoaxis signaling of Halobacterium salinarum

In Halobacterium salinarum phototaxis is mediated by the visual pigment-like photoreceptors sensory rhodopsin I (SRI) and II (SRII). SRI is a receptor for attractant orange and repellent UV-blue light, and SRII is a receptor for repellent blue-green light, and transmit signals through the membrane-bound transducer proteins HtrI and HtrII, respectively. ^ The primary sequences of HtrI and HtrII predict 2 transmembrane helices (TM1 and TM2) followed by a hydrophilic cytoplasmic domain. HtrII shows an additional large periplasmic domain for chemotactic ligand binding. The cytoplasmic regions are homologous to the adaptation and signaling domains of eubacterial chemotaxis receptors and, like their eubacterial homologs, modulate the transfer of phosphate groups from the histidine protein kinase CheA to the response regulator CheY that in turn controls flagellar motor rotation and the cell's swimming behavior. HtrII and Htrl are dimeric proteins which were predicted to contain carboxylmethylation sites in a 4-helix bundle in their cytoplasmic regions, like eubacterial chemotaxis receptors. ^ The phototaxis transducers of H. salinarum have provided a model for studying receptor/tranducer interaction, adaptation in sensory systems, and the role of membrane molecular complexes in signal transduction. ^ Interaction between the transducer HtrI and the photoreceptor SRI was explored by creating six deletion constructs of HtrI, with progressively shorter cytoplasmic domains. This study confirmed a putative chaperone-like function of HtrI, facilitating membrane insertion or stability of the SRI protein, a phenomenon previously observed in the laboratory, and identified the smallest HtrI fragment containing interaction sites for both the chaperone-like function and SRI photocycle control. The active fragment consisted of the N-terminal 147 residues of the 536-residue HtrI protein, a portion of the molecule predicted to contain the two transmembrane helices and the first ∼20% of the cytoplasmic portion of the protein. ^ Phototaxis and chemotaxis sensory systems adapt to stimuli, thereby signaling only in response to changes in environmental conditions. Observations made in our and in other laboratories and homologies between the halobacterial transducers with the chemoreceptors of enteric bacteria anticipated a role for methylation in adaptation to chemo- and photostimuli. By site directed mutagenesis we identified the methylation sites to be the glutamate pairs E265–E266 in HtrI and E513–E514 in HtrII. Cells containing the unmethylatable transducers are still able to perform phototaxis and adapt to light stimuli. By pulse-chase analysis we found that methanol production from carboxylmethyl group hydrolysis occurs upon specific photo stimulation of unmethylatable HtrI and HtrII and is due to turnover of methyl groups on other transducers. We demonstrated that the turnover in wild-type H. salinarum cells that follows a positive stimulus is CheY-dependent. The CheY-feedback pathway does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell. ^ Assembly of signaling molecules into architecturally defined complexes is considered essential in transmission of the signals. The spectroscopic characteristics of SRI were exploited to study the stoichiometric composition in the phototaxis complex SRI-HtrI. A molar ratio of 2.1 HtrI: 1 SRI was obtained, suggesting that only 1 SRI binding site is occupied on the HtrI homodimer. We used gold-immunoelectron microscopy and light fluorescence microscopy to investigate the structural organization and the distribution of other halobacterial transducers. We detected clusters of transducers, usually near the cell's poles, providing a ultrastructural basis for the global effects and intertransducer communication we observe. ^

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.

Three-dimensional structure of tropism-switching Bordetella bacteriophage.

Bacteriophage BPP-1, which infects Bordetella species, can switch its specificity by mutations to the ligand-binding surface of its major tropism-determinant protein, Mtd. This targeted mutagenesis results from the activity of a phage-encoded diversity-generating retroelement. Purified Mtd binds its receptor with low affinity, yet BPP-1 binding and infection of Bordettella cells are efficient because of high-avidity binding between phage-associated Mtd and its receptor. Here, using an integrative approach of three-dimensional (3D) structural analyses of the entire phage by cryo-electron tomography and single-prticle cryo-electron microscopy, we provide direct localization of Mtd in the phage and the structural basis of the high-avidity binding of the BPP-1 phage. Our structure shows that each BPP-1 particle has a T = 7 icosahedral head and an unusual tail apparatus consisting of a short central tail "hub," six short tail spikes, and six extended tail fibers. Subtomographic averaging of the tail fiber maps revealed a two-lobed globular structure at the distal end of each long tail fiber. Tomographic reconstructions of immuno-gold-labeled BPP-1 directly localized Mtd to these globular structures. Finally, our icosahedral reconstruction of the BPP-1 head at 7A resolution reveals an HK97-like major capsid protein stabilized by a smaller cementing protein. Our structure represents a unique bacteriophage reconstruction with its tail fibers and ligand-binding domains shown in relation to its tail apparatus. The localization of Mtd at the distal ends of the six tail fibers explains the high avidity binding of Mtd molecules to cell surfaces for initiation of infection.

CART: an Hrs/actinin-4/BERP/myosin V protein complex required for efficient receptor recycling.

Altering the number of surface receptors can rapidly modulate cellular responses to extracellular signals. Some receptors, like the transferrin receptor (TfR), are constitutively internalized and recycled to the plasma membrane. Other receptors, like the epidermal growth factor receptor (EGFR), are internalized after ligand binding and then ultimately degraded in the lysosome. Routing internalized receptors to different destinations suggests that distinct molecular mechanisms may direct their movement. Here, we report that the endosome-associated protein hrs is a subunit of a protein complex containing actinin-4, BERP, and myosin V that is necessary for efficient TfR recycling but not for EGFR degradation. The hrs/actinin-4/BERP/myosin V (CART [cytoskeleton-associated recycling or transport]) complex assembles in a linear manner and interrupting binding of any member to its neighbor produces an inhibition of transferrin recycling rate. Disrupting the CART complex results in shunting receptors to a slower recycling pathway that involves the recycling endosome. The novel CART complex may provide a molecular mechanism for the actin-dependence of rapid recycling of constitutively recycled plasma membrane receptors.

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

Aminoacyl-tRNA synthetases: Probing the molecular basis of catalysis and discrimination

Aminoacyl-tRNA synthetases (RSs) are responsible for the essential connection of amino acids with trinucleotide sequences of tRNA's. The RS family constitutes two structurally dissimilar groups of proteins, class I and class II. Methionyl-tRNA synthetase (MetRS) and isoleucyl-tRNA synthetase (IleRS), both members of class I, were the focus of this work. Both enzymes are zinc-containing proteins; show a high degree of amino acid specificity; and edit activated noncognate amino acids, thereby ensuring the fidelity of the genetic code. The goals of this work were to further delineate the molecular basis of catalysis and discrimination in these enzymes by mapping active site geometries using high-resolution nuclear magnetic resonance spectroscopy (NMR).^ Internuclear distances obtained from transferred nuclear Overhauser effects were used to define the conformations of Mg($\alpha$,$\beta$-methylene)ATP bound to E. coli MetRS and E. coli IleRS in multiple complexes. Identical conformations were found for the bound ATP. Thus, the predicted structural homology between IleRS and MetRS is supported by consensus enzyme-bound nucleotide conformations. The conformation of the bound nucleotide is not sensitive to occupation of the amino acid site of MetRS or IleRS. Therefore, conformational changes known to occur in the synthetases upon ligand binding appear not to alter the bound conformation of the adenosine portion of the nucleotide. Nuclear Overhauser effects on the substrate amino acid L-selenomethionine were also used to evaluate the enzyme-bound conformation of the cognate amino acid. The amino acid assumes a conformation which is consistent with a proposed editing mechanism.^ The E. coli MetRS was shown to catalyze amino acid $\alpha$-proton exchange in the presence of deuterium oxide of all cognate amino acids. It is proposed that the enzyme-bound zinc coordinates the $\alpha$-carboxylate of the amino acid, rendering the $\alpha$-proton more acidic. An enzymic base is responsible for exchange of the $\alpha$-proton. This proposal suggests that the enzyme-bound zinc may have a role in amino acid discrimination in MetRS. However, the role of this exchange reaction in catalysis remains unknown. ^

Structural and functional studies of the human integrin $\alpha 4\beta 1$ (CD49d/CD29)

The integrin receptor $\alpha 4\beta 1$ is a cell surface heterodimer involved in a variety of highly regulated cellular interactions. The purpose of this dissertation was to identify and characterize unique structural and functional properties of the $\alpha 4\beta 1$ molecule that may be important for adhesion regulation and signal transduction. To study these properties and to establish a consensus sequence for the $\alpha 4$ subunit, cDNA encoding $\alpha 4$ was cloned and sequenced. A comparison with previously described human $\alpha 4$ sequences identified several substitutions in the $5\prime$ and $3\prime$ untranslated regions, and a nonsynonymous G to A transition in the coding region, resulting in a glutamine substitution for arginine. Further analysis of this single nucleotide substitution indicated that two variants of the $\alpha 4$ subunit exist, and when compared with three ancestrally-related species, the new form cloned in our laboratory was found to be evolutionarily conserved.^ The expression of $\alpha 4$ cDNA in transfected K562 erythroleukemia cells, and subsequent studies using flow cytofluorometric, immunochemical, and ligand binding/blocking analyses, confirmed $\alpha 4\beta 1$ as a receptor for fibronectin (FN) and vascular cell adhesion molecule-1 (VCAM-1), and provided a practical means of identifying two novel monoclonal antibody (mAb) binding epitopes on the $\alpha 4\beta 1$ complex that may play important roles in the regulation of leukocyte adhesion.^ To investigate the association of $\alpha 4\beta 1$-mediated adhesion with signals involved in the spreading of lymphocytes on FN, a quantitative method of analysis was developed using video microscopy and digital imaging. The results showed that HPB-ALL $(\alpha 4\beta 1\sp{\rm hi},\ \alpha 5\beta 1\sp-)$ cells could adhere and actively spread on human plasma FN, but not on control substrate. Many cell types which express different levels of the $\alpha 4\beta 1$ and $\alpha 5\beta 1$ FN binding integrins were examined for their ability to function in these events. Using anti-$\alpha 4$ and anti-$\alpha 5$ mAbs, it was determined that cell adhesion to FN was influenced by both $\beta 1$ integrins, while cell spreading was found to be dependent on the $\alpha 4\beta 1$ complex. In addition, inhibitors of phospholipase A$\sb2$ (PLA$\sb2$), 5-lipoxygenases, and cyclooxygenases blocked HPB-ALL cell spreading, yet had no effect on cell adhesion to FN, and the impaired spreading induced by the PLA$\sb2$ inhibitor cibacron blue was restored by the addition of exogenous arachidonic acid (AA). These results suggest that the interaction of $\alpha 4\beta 1$ with FN, the activation of PLA$\sb2,$ and the subsequent release of AA, may be involved in lymphocyte spreading. ^

Mechanisms of TGF-beta1-induced neuronal plasticity in Aplysia

Transforming growth factor beta-1 (TGF-β1) is a cytokine and neurotrophic factor whose neuromodulatory effects in Aplysia californica were recently described. Previous results demonstrated that TGF-β1 induces long-term increases in the efficacy of sensorimotor synapses, a neural correlate of sensitization of the defensive tail withdrawal reflex. These results provided the first evidence that a neurotrophic factor regulates neuronal plasticity associated with a simple form of learning in Aplysia, and raised many questions regarding the nature of the modulation. No homologs of TGF-β had previously been identified in Aplysia, and thus, it was not known whether components of TGF-β1 signaling pathways were present in Aplysia. Furthermore, the signaling mechanisms engaged by TGF-β1 had not been identified, and it was not known whether TGF-β1 regulated other aspects of neuronal function.^ The present investigation into the actions of TGF-β1 was initiated by examining the distribution of the type II TGF-β1 receptor, the ligand binding receptor. The receptor was widely distributed in the CNS and most neurons exhibited somatic and neuritic immunoreactivity. In addition, the ability of TGF-β1 to activate the cAMP/PKA and MAPK pathways, known to regulate several important aspects of neuronal function, was examined. TGF-β1 acutely decreased cAMP levels in sensory neurons, activated MAPK and triggered translocation of MAPK to the nucleus. MAPK activation was critical for both short- and long-term regulation of neuronal function by TGF-β1. TGF-β1 acutely decreased synaptic depression induced by low frequency stimuli in a MAPK-dependent manner. This regulation may result, at least in part, from the modulation of synapsin, a major peripheral synaptic vesicle protein. TGF-β1 stimulated MAPK-dependent phosphorylation of synapsin, a process believed to regulate synaptic vesicle mobilization from reserve to readily-releasable pools of neurotransmitter. In addition to its acute effect on synaptic efficacy, TGF-β1 also induced long-term increases in sensory neuron excitability. Whereas transient exposure to TGF-β1 was not sufficient to drive short-or long-term changes in excitability, prolonged exposure to TGF-β1 induced long-term changes in excitability that depended on MAPK. The results of these studies represent significant progress toward an understanding of the role of TGF-β1 in neuronal plasticity. ^

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Spectroscopic and functional investigations of the AMPA subtype of ionotropic glutamate receptors

Ion channels play a crucial role in the functioning of different systems of the body because of their ability to bridge the cell membrane and allow ions to pass in and out of the cell. Ionotropic glutamate receptors are one class of these important proteins and have been shown to be critical in propagating synaptic transmission in the central nervous system and in other diverse functions throughout the body. Because of their wide-ranging effects, this family of receptors is an important target for structure-function investigations to understand their mechanism of action. ^ α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one subtype of glutamate receptors and have been shown to be the primary receptors involved in rapid excitatory signaling in the central nervous system. Agonist binding to the extracellular ligand binding domain of these receptors causes various conformational changes that culminate in formation of the ion channel. Previous structural investigations have provided important information about their mechanism of action, including uncovering a relationship between the degree of cleft closure in the binding domain and activation of the receptor. However, what question remains unanswered is how specific interactions between the agonist and the protein interplay with cleft closure to mediate receptor activation. ^ To investigate this question, I applied a multiscale approach to investigate the effects of agonist binding on various levels. Vibrational spectroscopy was utilized to investigate molecular-level interactions in the binding pocket, and fluorescence resonance energy transfer (FRET) was employed to measure cleft closure in the isolated ligand binding domain. The results of these studies in the isolated binding domain were then correlated to activation of the full receptor. These investigations showed a relationship between the strength of the interaction at the α-amine group of the agonist and extent of receptor activation, where a stronger interaction correlated to a larger activation, which was upheld even when the extent of cleft closure did not correlate to activation. These results show that this interaction at the α-amine group is critical in mediating the allosteric mechanism of activation and provide a bit more insight into how agonist binding is coupled to channel gating in AMPA receptors. ^

Organization and function of platelet glycoprotein Ib-IX complex

Glycoprotein (GP) Ib-IX complex, the second most abundant receptor expressed on the platelet surface, plays critical roles in haemostasis and thrombosis by binding to its ligand, von Willebrand factor (vWF). Defect or malfunction of the complex leads to severe bleeding disorders, heart attack or stroke. Comprised of three type I transmembrane subunits—GPIbα, GPIbβ and GPIX, efficient expression of the GPIb-IX complex requires all three subunits, as evident from genetic mutations identified in the patients and reproduced in transfected Chinese hamster ovary (CHO) cells. However, how the subunits are assembled together and how the complex function is regulated is not fully clear. By probing the interactions among the three subunits in transfected cells, we have demonstrated that the transmembrane domains of the three subunits interact with one another, facilitating formation of the two membrane-proximal disulfide bonds between GPIbα and GPIbβ. We have also identified the interface between extracellular domains of GPIbβ and GPIX, and provided evidence suggesting a direct interaction between extracellular domains of GPIbα and GPIX. All of these interactions are not only critical for correct assembly and consequently efficient expression of the GPIb-IX complex on the cell surface, but also for its function, such as the proper ligand binding, since removing the two inter-subunit disulfide bonds significantly hampers vWF binding to the complex under both static and physiological flow conditions. The two inter-subunit disulfide bonds are also critical for regulating the ectodomain shedding of GPIbα by the GPIbβ cytoplasmic domain. Mutations in the juxtamembrane region of the GPIbβ cytoplasmic domain deregulate GPIbα shedding, and such deregulation is further enhanced when the two inter-subunit disulfide bonds are removed. In summary, we have established the overall organization of the GPIb-IX complex, and the importance of proper organization on its function. ^