The role of Cysteine 6.47 in class A GPCRs

Abstract Background: The CWxP motif of transmembrane helix 6 (x: any residue) is highly conserved in class A GPCRs. Within this motif, W6.48 is a big star in the theory of the global “toggle switch” because of its key role in the activation mechanism of GPCRs upon ligand binding. With all footlights focused on W6.48, the reason why the preceding residue, C6.47, is largely conserved is still unknown. The present study is aimed to fill up this lack of knowledge by characterizing the role of C6.47 of the CWxP motif. Results: A complete analysis of available crystal structures has been made alongside with molecular dynamics simulations of model peptides to explore a possible structural role for C6.47. Conclusions: We conclude that C6.47 does not modulate the conformation of the TM6 proline kink and propose that C6.47 participates in the rearrangement of the TM6 and TM7 interface accompanying activation.

Mécanismes moléculaires d’activation du récepteur A des peptides natriurétiques

Le récepteur A des peptides natriurétiques (NPRA) fait partie de la famille des guanylates cyclases membranaires. L’activation du NPRA par ses agonistes naturels, ANP et BNP, induit une production de GMPc qui est responsable de leur rôle dans l’homéostasie cardiovasculaire, l’inhibition de l’hypertrophie et de la fibrose cardiaques et la régulation de la lipolyse. Le NPRA est un homodimère non covalent composé d’un domaine extracellulaire de liaison du ligand (ECD), d’un unique domaine transmembranaire (TM), d’un domaine d’homologie aux kinases et d’un domaine guanylate cyclase. Bien que le NPRA ait un rôle physiologique important, les mécanismes moléculaires régissant son processus d’activation restent inconnus. Nous avons donc analysé les premières étapes du processus d’activation du NPRA. Nous avons d'abord étudié le rôle de la dimérisation des ECD dans l’activation du récepteur. Nous avons utilisé les techniques de liaison de radioligand, de FRET et de modélisation moléculaire, pour caractériser la liaison à l’ECD des agonistes naturels, d’un superagoniste et d’un antagoniste. L’ANP se lie à un dimère d’ECD préformé et la dimérisation spontanée est l’étape limitante du processus de liaison. De plus, comme le démontrent nos études de FRET, tous les peptides, incluant l’antagoniste, stabilisent le récepteur sous sa forme dimérique. Cependant, l’antagoniste A71915 stabilise le dimère d’ECD dans une conformation différente de celle induite par l’ANP. La dimérisation du NPRA semble donc nécessaire, mais non suffisante à l’activation du récepteur. L’état d’activation du NPRA dépend plutôt de l’orientation des sous unités dans le dimère. Nous avons ensuite étudié le mécanisme moléculaire de transduction du signal à travers la membrane. Plusieurs études ont suggéré que l’activation du NPRA implique un changement de conformation du domaine juxtamembranaire (JM). Cependant, les études de cristallographie de l’ECD soluble de NPRA n’ont pas permis de documenter la structure du JM et le changement de conformation impliqué dans la transduction du signal reste inconnu. Pour analyser ce changement de conformation, nous avons d’abord séquentiellement substitué les neuf acides aminés du JM par une cystéine. En étudiant la capacité des mutants à former des dimères covalents de façon constitutive ou induite par l’ANP, nous avons pu évaluer la proximité relative des résidus du JM, avant et après activation du NPRA. Ces résultats ont démontré la proximité élevée de certains résidus spécifiques et sont en contradiction avec les données cristallographiques. Nous avons également démontré que le domaine intracellulaire impose une contrainte conformationnelle au JM à l’état de base, qui est levée après liaison de l’ANP. En introduisant de 1 à 5 alanines dans l’hélice-α transmembranaire, nous avons montré qu’une rotation des TM de 40° induit une activation constitutive du NPRA. Le signal d’activation pourrait donc être transmis à travers la membrane par un mécanisme de rotation des TM. En utilisant nos données expérimentales, nous avons généré le premier modèle moléculaire illustrant la conformation active du NPRA, où les domaines JM et TM sont représentés. Dans son ensemble, cette étude apporte une meilleure compréhension des mécanismes moléculaires régissant les premières étapes du processus complexe d’activation du NPRA.

Peptide inhibitors of G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) are regulatory enzymes involved in the modulation of seven-transmembrane-helix receptors. In order to develop specific inhibitors for these kinases, we synthesized and investigated peptide inhibitors derived from the sequence of the first intracellular loop of the beta(2)-adrenergic receptor. Introduction of changes in the sequence and truncation of N- and C-terminal amino acids increased the inhibitory potency by a factor of 40. These inhibitors not only inhibited the prototypical GRK2 but also GRK3 and GRK5. In contrast there was no inhibition of protein kinase C and protein kinase A even at the highest concentration tested. The peptide with the sequence AKFERLQTVTNYFITSE inhibited GRK2 with an IC50 of 0.6 mu M, GRK3 with 2.6 mu M and GRK5 with 1.6 mu M. The peptide inhibitors were non-competitive for receptor and ATP. These findings demonstrate that specific peptides can inhibit GRKs in the submicromolar range and suggest that a further decrease in size is possible without losing the inhibitory potency. (c) 2005 Published by Elsevier Inc.

New molecular variants of hypothalamus-pituitary-gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore)

Endocrine system plays a major role in the control of reproductive functions which are regulated by the hypothalamus-pituitary-gonad axis and its interactions. FSH and LH receptor genes are expressed at the gonads and GnRH receptor gene is expressed at the anterior pituitary gland. Misense mutations of the FSH, LH or GnRH receptors, activating or inactivating their functions in mammals, are potentially useful to allow the understanding of the role of this group of gonadotropins in reproductive phenotypes as early puberty and birth interval length. In the present study, polymorphisms in bovine exon 11 and 3`UTR of LHR, exon 10 and 3`UTR of FSHR and GnRHR genes were characterized with some of them resulting in changes in the aminoacidic chain. These polymorphic sites were found in a Bos taurus indicus (Nellore) female population by means of PCR-SSCP and DNA sequencing. Association between nucleotidic/aminoacidic changes and early puberty were determined by Chi-square analysis. It was found association between FSHR 3`UTR polymorphisms at position 2181, 2248 and 2249 bp and early puberty phenotype (p < 0.05). The presence of these new molecular markers might be considered in further studies to validate its correlation with early puberty or other reproduction associated phenotypes in cattle breeds. (C) 2007 Published by Elsevier B.V.

Untersuchung der Struktur und Assemblierung des Lichtsammelkomplexes II höherer Pflanzen mittels elektronenparamagnetischer Resonanz (EPR)

Der Haupt-Lichtsammelkomplex (LHCII) des Photosyntheseapparates höherer Pflanzen gehört zu den häufigsten Membranproteinen der Erde. Seine Kristallstruktur ist bekannt. Das Apoprotein kann rekombinant in Escherichia coli überexprimiert und somit molekularbiologisch vielfältig verändert werden. In Detergenzlösung besitzt das denaturierte Protein die erstaunliche Fähigkeit, sich spontan zu funktionalen Protein-Pigment-Komplexen zu organisieren, welche strukturell nahezu identisch sind mit nativem LHCII. Der Faltungsprozess findet in vitro im Zeitbereich von Sekunden bis Minuten statt und ist abhängig von der Bindung der Cofaktoren Chlorophyll a und b sowie verschiedenen Carotinoiden.rn Diese Eigenschaften machen LHCII besonders geeignet für Strukturuntersuchungen mittels der elektronenparamagnetischen Resonanz (EPR)-Spektrokopie. Diese setzt eine punktspezifische Spinmarkierung des LHCII voraus, die in dieser Arbeit zunächst optimiert wurde. Einschließlich der Beiträge Anderer stand eine breite Auswahl von über 40 spinmarkierten Mutanten des LHCII bereit, einen N-terminalen „Cys walk“ eingeschlossen. Weder der hierfür notwendige Austausch einzelner Aminosäuren noch die Anknüpfung des Spinmarkers beeinträchtigten die Funktion des LHCII. Zudem konnte ein Protokoll zur Präparation heterogen spinmarkierter LHCII-Trimere entwickelt werden, also von Trimeren, die jeweils nur ein Monomer mit einer Spinmarkierung enthalten.rn Spinmarkierte Proben des Detergenz-solubilisierten LHCII wurden unter Verwendung verschiedener EPR-Techniken strukturell analysiert. Als besonders aussagekräftig erwies sich die Messung der Wasserzugänglichkeit einzelner Aminosäurepositionen anhand der Electron Spin Echo Envelope Modulation (ESEEM). In Kombination mit der etablierten Double Electron-Electron Resonance (DEER)-Technik zur Detektion von Abständen zwischen zwei Spinmarkern wurde der membranständige Kernbereich des LHCII in Lösung eingehend untersucht und strukturell der Kristallstruktur für sehr ähnlich befunden. Die Vermessung kristallographisch nicht erfasster Bereiche nahe dem N-Terminus offenbarte die schon früher detektierte Strukturdynamik der Domäne in Abhängigkeit des Oligomerisierungsgrades. Der neue, noch zu vervollständigende Datensatz aus Abstandsverteilungen und ESEEM-Wasserzugänglichkeiten monomerer wie trimerer Proben sollte in naher Zukunft die sehr genaue Modellierung der N-terminalen Domäne des LHCII ermöglichen.rn In einem weiteren Abschnitt der Arbeit wurde die Faltung des LHCII-Apoproteins bei der LHCII-Assemblierung in vitro untersucht. Vorausgegangene fluoreszenzspektroskopi-sche Arbeiten hatten gezeigt, dass die Bindung von Chlorophyll a und b in aufeinanderfolgenden Schritten im Zeitbereich von weniger als einer Minute bzw. mehreren Minuten erfolgten. Sowohl die Wasserzugänglichkeit einzelner Aminosäurepositionen als auch Spin-Spin-Abstände änderten sich in ähnlichen Zeitbereichen. Die Daten deuten darauf hin, dass die Ausbildung der mittleren Transmembran-Helix mit der schnelleren Chlorophyll-a-Bindung einhergeht, während sich die Superhelix aus den beiden anderen Transmembranhelices erst im langsameren Schritt, zusammen mit der Chlorophyll-b-Bindung, ausbildet.rn

Funktion der C 4-Dicarboxylat-Transporter DctA und DcuB als Co-Sensoren von DcuS in Escherichia coli

Escherichia coli kann C4-Dicarboxylate sowohl unter aeroben als auch unter anaeroben Bedingungen zur Energiekonservierung nutzen. Die Synthese der beteiligten Transporter und Enzyme wird auf der Transkriptionsebene durch das Zweikomponentensystem DcuSR reguliert. DcuS ist der Sensor für C4-Dicarboxylate. Der Antwortregulator DcuR wird von DcuS aktiviert und induziert die Expression des C4-Dicarboxylat-Transporters DctA unter aeroben Verhältnissen. Anaerob verstärkt DcuSR die Expression des Fumarat/Succinat-Antiporters DcuB, der Fumarase B und der Fumaratreduktase FrdABCD. DctA und DcuB agieren als Co-Sensoren von DcuS und üben einen negativen Effekt auf die Genexpression von dctA bzw. dcuB aus.rnIn dieser Arbeit wurde die Funktion von DctA und DcuB als Co-Sensoren von DcuS untersucht. Sowohl für DcuB als auch für DctA wurde eine direkte Protein-Protein-Interaktion mit DcuS über ein bakterielles Two-Hybrid System nachgewiesen. DcuS bildete ein Transporter-Sensor-Cluster mit DctA und DcuB. C-terminale Verkürzung und die Mutagenese einzelner Aminosäuren der C-terminalen Helix 8b von DctA führten zu einem Verlust der Interaktion mit DcuS. Mit dieser Interaktion gingen sowohl die regulatorische Funktion als auch die Transportfunktion der Punktmutante DctA-L414A verloren. Ein Verlust der Interaktion wurde ebenfalls zwischen einer konstitutiv aktiven DcuS-Mutante und wildtypischem DctA beobachtet. Ebenso zeigte sich eine partielle Reduktion der Interaktion von DcuS mit DctA, wenn DcuS nach der zweiten Transmembranhelix verkürzt wurde. Die Interaktion zwischen DcuS und DctA wurde durch den Effektor Fumarat modifiziert, ging aber nicht komplett verloren.rnDctA konnte in verschiedenen Plasmidsystemen überproduziert werden und bildete Homotrimere. Die Topologie von DctA wurde mit experimentellen und in silico Methoden aufgeklärt. DctA ähnelt der Struktur und Topologie des Aminosäuretransporters Glt aus Pyrococcus horikoshii. DctA besitzt acht Transmembranhelices mit einem cytosolischen N- und C-Terminus sowie zwei Haarnadelschleifen. Die Substratbindung findet höchstwahrscheinlich in den Haarnadelschleifen statt und der Transport erfolgt nach dem „alternating access“ Modell.rnAußerdem wurde die Funktion des Transporters YfcC untersucht. Das Gen yfcC wurde mit Schlüsselgenen des Acetatstoffwechsels co-transkribiert. In yfcC-Deletionsstämmen zeigte sich ein stammspezifischer Defekt bei Wachstum mit Acetat und Transport von Acetat.

Einfluss membranmimetischer Umgebungen auf die Dimerisierung von Glykophorin A

Bei der Untersuchung von Membranproteinen bedarf es der Entwicklung von neuen Methoden, da Standardmethoden, entwickelt für lösliche Proteine, meist nicht auf Membranproteine angewendet werden können. Das größte Problem besteht in der schlechten Wasserlöslichkeit der Membranproteine, da diese sich in vivo in einer hydrophoben Umgebung, der Membran, befinden. Um dennoch isolierte Membranproteine und ihre Faltung in vitro charakterisieren zu können, sind membranmimetische Systeme notwendig um Membranproteine in Lösung zu bringen. In dieser Arbeit wurden Lysophosphocholin Detergenzien, die Copolymere Amphipol A8-35, p(HMPA)-co-p(LMA) sowie synthetische Membranen aus Phospholipiden auf Ihre Eigenschaften in wässriger Lösung untersucht, und deren Auswirkungen auf die Solubilisierung und Dimerisierung der Glykophorin A (GpA)-Transmembranhelix analysiert. Es wurde erstmals gezeigt, dass die Aggregtionszahl von Detergenzmizellen die Dimerisierung von GpA beeinflusst. Die Copolymere A8-35 und pHPMA-pLMA sind in der Lage die Sekundärstruktur von GpA sowie dessen Dimer zu stabilisieren. Allerdings ist dies bei pHPMA-pLMA Copolymeren erst ab einem LMA-Anteil von über 15% möglich. In synthetischen Membranen zeigte die Dimerisierung von GpA eine Abhängigkeit von negativ geladenen Lipiden, die die Dimerisierung zwar vermindern aber die Ausbildung der Transmembranhelix fördern. Eine Zugabe von physiologischen Konzentrationen an Calciumionen ändert die Membraneigenschaften drastisch aber die Dimerisierung von GpA wird nur geringfügig beeinflusst.

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.

Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Functional analysis of GNA -1, a Galphai superfamily member found in the filamentous fungus Neurospora crassa

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cAMP phosphodiesterase, phospolipase C-β, mitogen-activated protein kinases, and ion channels. ^ Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric Gα proteins (GNA-1–3) and one Gβ subunit (GNB-1). GNA-1 was the first microbial protein that could be classified in the Gαi superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. ^ Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The Gαi and Gαz members of the Gαi superfamily negatively regulate adenylyl cyclase activity in mammalian cells; therefore, adenylyl cyclase and cAMP levels were measured in Δgna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second Gα in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. ^ Genetic and biochemical epistasis experiments utilizing a Δ gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and crude membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Δgna-1 membrane fractions. These studies provide direct evidence for the involvement of Gα proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes. ^

Heterotrimeric G protein -mediated signal transduction in Neurospora crassa

Heterotrimeric G protein-mediated signal transduction is one of numerous means that cells utilize to respond to external stimuli. G proteins consist of α, β andγ subunits. Extracellular ligands bind to seven-transmembrane helix receptors, triggering conformational changes. This is followed by activation of coupled G proteins through the exchange of GDP for GTP on the Gα subunit. Once activated, Gα-GTP dissociates from the βγ dimer. Both of these two moieties can interact with downstream effectors, such as adenylyl cyclase, phospholipase C, phosphodiesterases, or ion channels, leading to a series of changes in cellular metabolism and physiology. ^ Neurospora crassa is a eukaryotic multicellular filamentous fungus, with asexual/vegetative and sexual phases to its life cycle. Three Gα (GNA-1, GNA-2, GNA-3) and one Gβ (GNB-1) proteins have been identified in this organism. This dissertation investigates GNA-1 and GNB-1 mediated signaling pathways in N. crassa. ^ GNA-1 was the first identified microbial Gα that belongs to a mammalian superfamily (Gαi). Deletion of GNA-1 leads to multiple defects in N. crassa. During the asexual cycle, Δgna-1 strains display a slower growth rate and delayed conidiation on solid medium. In the sexual cycle, the Δgna-1 mutant is male-fertile but female-sterile. Biochemical studies have shown that Δ gna-1 strains have lower adenosine 3′–5 ′ cyclic monophosphate (cAMP) levels than wild type under conditions where phenotypic defects are observed. In this thesis work, strains containing one of two GTPase-deficient gna-1 alleles (gna-1 R178C, gna-1Q204L) leading to constitutive activation of GNA-1 have been constructed and characterized. Activation of GNA-1 causes uncontrolled aerial hyphae proliferation, elevated sensitivity to heat and oxidative stresses, and lower carotenoid synthesis. To further study the function of GNA-1, constructs to enable expression of mammalian Gαi superfamily members were transformed into a Δ gna-1 strain, and complementation of Δgna-1 defects investigated. Gαs, which is not a member of Gα i superfamily was used as a control. These mammalian Gα genes were able to rescue the vegetative growth rate defect of the Δ gna-1 strain in the following order: Gαz > Gα o > Gαs > Gαt > Gαi. In contrast, only Gαo was able to complement the sexual defect of a Δgna-1 strain. With regard to the thermotolerance phenotype, none of the mammalian Gα genes restored the sensitivity to a wild type level. These results suggest that GNA-1 regulates two independent pathways during the vegetative and sexual cycles in N. crassa. ^ GNB-1, a G protein β subunit from N. crassa, was identified and its functions investigated in this thesis work. The sequence of the gnb-1 gene predicts a polypeptide of 358 residues with a molecular mass of 39.7 kDa. GNB-1 exhibits 91% identity to Cryphonectria parasitica CPGB-1, and also displays significant homology with human and Dictyostelium Gβ genes (∼66%). A Δ gnb-1 strain was constructed and shown to exhibit defects in asexual spore germination, vacuole number and size, mass accumulation and female fertility. A novel role for GNB-1 in regulation of GNA-1 and GNA-2 protein levels was also demonstrated. ^

Constitutively active cAMP receptor mutants dominantly inhibit receptor-mediated signaling pathways in Dictyostelium discoideum

Dictyostelium, a soil amoeba, is able to develop from free-living cells to multicellular fruiting bodies upon starvation using extracellular cAMP to mediate cell-cell communication, chemotaxis and developmental gene expression. The seven transmembrane G protein-coupled cAMP receptor-1 (cAR1) mediated responses, such as the activation of adenylyl cyclase and guanylyl cyclase, are transient, due to the existence of poorly understood adaptation mechanisms. For this dissertation, the powerful genetics of the Dictyostelium system was employed to study the adaptation mechanism of cAR1-mediated cAMP signaling as well as mechanisms intrinsic to cAR1 that regulate its activation. ^ We proposed that constitutively active cAR1 would cause constant adaptation, thus inhibiting downstream pathways that are essential for aggregation and development. Therefore, a screen for dominant negative cAR1 mutants was undertaken to identify constitutively active receptor mutants. Three dominant negative cAR1 mutants were identified. All appear to be constitutively active receptor mutants because they are constitutively phosphorylated and possess high affinity for cAMP. Biochemical studies showed that these mutant receptors prevented the activation of downstream effectors, including adenylyl and guanylyl cyclases. In addition, these cells also were defective in cAMP chemotaxis and cAR1-mediated gene expression. These findings suggest that the mutant receptors block development by constantly activating multiple adaptation pathways. ^ Sequence analysis revealed that these mutations (I104N, L100H) are clustered in a conserved region of the third transmembrane helix (TM3) of cAR1. To investigate the role of this region in receptor activation, one of these residues, I104, was mutated to all the other 19 possible amino acids. We found that all but the most conservative substitutions increase the receptor's affinity about 20- to 70-fold. However, only highly polar substitutions of I104, particularly basic residues, resulted in receptors that are constitutively phosphorylated and dominantly inhibit development, suggesting that highly polar substitutions not only disrupt an interaction constraining the receptor in its low-affinity, inactive state but also promote an additional conformational change that resembles the ligand-bound conformation. Our findings suggest that I104 plays a specific role in constraining the receptor in its inactive state and that substituting it with highly polar residues results in constitutive activation. ^