<p>Seven transmembrane spanning receptors (7TMRs), or G-protein coupled receptors (GPCRs), represent the largest and most ubiquitous of the several families of plasma membrane receptors and regulate virtually all known physiological processes in humans. The classical paradigm of signal transduction in response to 7TMR stimulation involves an agonist-induced conformational change of the receptor which leads to interaction with and dissociation of the heterotrimeric G-protein into independent Galpha and Gbeta;gamma signaling subunits. Following their activation, 7TMRs are phosphorylated by G-protein coupled receptor kinases (GRKs) and subsequently recruit beta-arrestins. beta-arrestins are multifunctional adaptor proteins which not only desensitize G-protein signals, but also facilitate receptor internalization and mediate numerous signaling pathways on their own. As beta-arrestins universally interact with members of the 7TMR superfamily, we (1) developed an in vitro model system to assess conformational changes that occur in beta-arrestins in response to phosphorylation and (2) to map the sites of phosphorylation on the beta2 adrenergic receptor by different GRKs which would determine the conformation(s) assumed by beta-arrestin and thereby, in turn, instruct its functional capabilities. </p><p>We determined conformational changes in beta-arrestin1 in vitro using limited tryptic proteolysis and MALDI-TOF MS analysis in the presence of a phosphopeptides derived from the C-terminus of the V2 vasopressin receptor (V2Rpp or V2R4p) or the corresponding unphosphorylated peptide (V2Rnp). Upon V2Rpp binding, we show that the previously shielded R393 becomes accessible, which indicates release of the C-terminus. Moreover, we have shown that R285 becomes more accessible and this residue is located in a region of &beta;-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1. We also show a functional consequence of the release of beta-arrestin1's C-terminus by enhanced clathrin binding. In addition, we have shown marked protection of beta-arrestin1's N-domain in the presence of V2Rpp; consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. Using a differentially phsophorylated V2R peptide (V2R4p), we show that beta-arrestin1 is able to adopt distinct conformations in response to different phosphorylation patterns. Futhermore, a striking difference is observed in the conformation of V2Rpp-bound beta-arrestin1 when compared to beta-arrestin2, namely the flexibility of the inter-domain hinge region. These data represent the first direct evidence that the beta-arrestin1 conformation is differentially instructed by phosphorylation patterns and that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.</p><p>Phosphorylation of 7TMRs by GRKs plays essential roles in regulation of receptor function by promoting interactions of the receptors with beta-arrestins. We hypothesized that different GRKs phosphorylate distinct sets of sites thereby establishing a "bar code." In order to test this hypothesis, we monitored the phosphorylation events of the beta2AR upon stimulation with a classical full agonist, isoproterenol, or a beta-arrestin "biased" agonist, carvedilol, in the presence of a full complement of GRKs or when individual GRKs (2 or 6) were depleted by siRNA. We demonstrate that at least thirteen sites on the beta2AR show changes in phosphorylation in response to the agonist isoproterenol. Of these, phosphorylation increased 10 to more than 300 fold in 12 (S261, S262, S345, S346, S355, S356, T360, S364, S396, S401, S407 AND S411) and decreased 50% in one (S246). Depletion of GRK2 or 6 by siRNA indicates that S355, 356 are GRK6 sites whereas the remainder are GRK2 sites. Phosphorylation of GRK2 sites inhibits that of GRK6 sites. Carvedilol, a beta-arrestin biased agonist, promotes phosphorylation of only the GRK6 sites S355, 356. In HEK293 cells, GRK2 phosphorylation is found to be the major positive regulator of receptor internalization; to contribute to receptor desensitization; and to inhibit beta-arrestin mediated ERK activation. Phosphorylation of the two GRK6 sites contributes to receptor desensitization and internalization and is required for beta-arrestin mediated ERK activation. These data indicate that different ligands promote distinct patterns of receptor phosphorylation which dictate different patterns of beta-arrestin mediated function.</p> / Dissertation

<p>Sonic hedgehog (Shh) is a critical secreted signaling molecule that regulates many aspects of organogenesis. In the absence of Shh, many organs, including the foregut, larynx, palate, cerebellum and heart do not form properly. However, the cellular details of the roles of Shh, including the relevant domains of Shh expression and reception, have not been elucidated for many of these processes. </p><p>The single embryonic foregut tube must divide into the trachea and esophagus, which does not occur in the Shh-null mutant. In Chapter 5, I use Cre-Lox technology to determine that the ventral foregut endoderm is the relevant source of Shh for this process and the mesoderm must directly receive that Shh signal. Surprisingly, this signaling event appears to occur two days before the foregut begins to divide, indicating an early essential role for Shh in foregut division. </p><p>Shh is also expressed at later stages in the maturing trachea and esophagus. In Chapter 6, I demonstrate that these domains serve to establish differentiated mesoderm. In the trachea, Shh from the endoderm signals directly to the mesoderm to form the tracheal cartilage rings. In the esophagus, the roles of Shh are more complex. Shh regulates the size of the esophagus and controls patterning of the concentric rings of esophageal mesoderm, however this process seems to be indirect, requiring autocrine Shh signaling within the esophageal endoderm. </p><p>The laryngeal apparatus is entirely absent in the Shh-null mouse. I n Chapter 3, I dissect the domains of Shh expression and reception required for laryngeal development and demonstrate that loss of endodermal Shh expression causes laryngotracheoesophageal clefts and malformed laryngeal cartilages. As much of laryngeal morphogenesis poorly understood, I also utilize dual mesodermal and neural crest fate maps to determine the embryonic origins of various laryngeal tissues. Finally, as Shh signaling often occurs in concert with Bone Morphogenic Protein (BMP) signaling, I investigate the roles of BMP signaling in laryngeal development. </p><p>Much of Shh signaling occurs at the primary cilium, to which Smoothened, a critical pathway member, must translocate upon Shh signal transduction. This process requires a Smo-Kif3a-&#946;arretin complex in mammalian cell culture. However, the roles of &#946;arrestins in mouse development, and their relationship to Shh signaling have not been investigated in vivo. To do so, in Chapter 4, I analyze the phenotypes of the &#946;arr1/&#946;arr2 double knockout embryos and demonstrate that they have palatal, cerebellar, cardiovascular and renal defects consistent with a specific impairment of mitogenic Shh signaling. </p><p>Altogether, my work dissects the cellular details of Shh signaling during multiple organ systems in the mouse embryo. I further analyze the consequences of absent or misregulated Shh signaling across multiple developmental contexts and determine that Shh plays critical and diverse roles in organogenesis.</p> / Dissertation

The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system, that has been suggested as a target for the treatment of various disorders, including anxiety, pain and neurodegeneration. Despite the wide therapeutic potential of CB1, development of potential drug candidates has long been hindered by concerns about adverse effects, rapid tolerance development and abuse potential. Ligands that produce biased signaling have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. Biased signaling involves selective activation of a signaling transducer in detriment of another, mainly involving selective activation of G-protein signaling or b-arrestin signaling. However, biased signaling at the CB1 receptor is poorly understood due to the lack of strongly biased agonists. The development of biased agonists would be aided by understanding the molecular mechanism that leads to biased signaling. Although the structure of CB1 has been resolved in the inactive state and in the canonical active state, which allows G-protein signaling, little is known about the alternative active state that allows b-arrestin biased signaling. Therefore, we set out to investigate molecular and pharmacological tools that could shed light on the mechanism of CB1 biased signaling and to characterize novel allosteric ligands with a biased signaling profile. Using molecular dynamics stimulation of CB1 bound to a ORG27569, an allosteric ligand that stimulates b-arrestin signaling and inhibits G-protein signaling, we proposed single amino acid mutations that were predicted to impact b-arrestin signaling, and expressed wild-type and mutated CB1 receptor in HEK293 cells to measure signaling through different signaling transducers. We found that N7.49 and Y7.53, two amino acids in the highly conserved NPXXY motif, were essential for b-arrestin recruitment and signaling, but mutating them to Ala and Phe, respectively, did not impact G-protein signaling. We also found that I2.43, a functionally conserved amino acid on transmembrane helix 2, negatively regulates a switch in the rotameric position of Y7.53, as mutating I2.43 to Ala reduced steric hindrance upon Y7.53 and enhanced b-arrestin1 recruitment and signaling, while mutating it to Thr, a polar residue that would further hinder Y7.53, partially inhibited b-arrestin recruitment. Therefore, we concluded that N7.49 and Y7.53 form a hydrogen bond network along with D2.50 that is essential for the alternative active state that stimulates b-arrestin biased signaling. N7.49 acts as a fulcrum on which transmembrane helix 7 can bend, and Y7.53 acts as a rotamer toggle switch, stabilizing conformational changes on the intracellular end of transmembrane helix 7. This is the first record of a molecular mechanism for CB1 b-arrestin biased signaling involving the NPXXY motif. Due to the highly conserved character of these residues, it is possible that this mechanism can also be applied to other class A G-protein coupled receptors. In addition, we characterized novel biased allosteric ligands that stimulate or inhibit b-arrestin1 signaling. Two ORG27569 analogs were found to enhance orthosteric agonist induced b-arrestin1 recruitment and extracellular-signal regulated kinase 1/2 phosphorylation (pERK), with no effect on G-protein signaling. Two pregnenolone analogs absent of the steroid scaffold were found to inhibit pERK signaling independent of Gprotein signaling, indicating that they hinder b-arrestin dependent signaling. Since these analogs are believed to mediate their effects via stimulation or inhibition of conformational changes on transmembrane helix 7, our findings support a role for this domain on the alternative active state of CB1. In contrast, a GAT211 analog, GAT1601, had no effect on recruitment of b-arrestin1, but stimulated G-protein signaling and slightly enhanced barrestin2 recruitment. This compound binds to an allosteric site, where it stimulates the canonical active state of CB1 by facilitating the outward movement of transmembrane helix 6. Altogether, the results presented in this dissertation suggest that CB1 b-arrestin biased signaling is regulated by the NPXXY motif, which stimulates conformational changes on the transmembrane helix 7/helix 8 elbow, and that stimulating or hindering these conformational changes can enhance or disrupt CB1 b-arrestin biased signaling. However, facilitating the movement of transmembrane helix 6 favors G-protein biased signaling. Our findings provide molecular and pharmacological tools that will be of great importance to structure guided drug design and to future studies on the functional consequences of biased signaling at the CB1 receptor.

No description available.

CB1 receptors (CB1R) mediate the psychoactive and therapeutic effects of cannabinoids including ∆9-tetrahydrocannabinol (THC), the main psychoactive constituent in marijuana. However, therapeutic use is limited by side effects and tolerance and dependence with chronic administration. Tolerance to cannabinoid-mediated effects is associated with CB1R adaptations, including desensitization (receptor-G-protein uncoupling) and downregulation (receptor degradation). The objectives of this thesis are to investigate the regional-specificity in CB1R function and regulation. Previous studies have investigated CB1Rs in a subset of regions involved in cannabinoid effects, but an inclusive regional comparison of the relative efficacies of different classes of cannabinoids to activate G-proteins has not been conducted. A novel unbiased whole-brain analysis was developed based on Statistical Parametric Mapping (SPM) for 3D-reconstructed mouse brain images derived from agonist-stimulated [35S]GTPgS autoradiography, which has not been described before. SPM demonstrated regional differences in the relative efficacies of cannabinoid agonists methanandamide (M-AEA), CP55,940 (CP), and WIN55,212-2 (WIN) in mouse brains. To assess potential contribution of novel sites, CB1R knockout (KO) mice were used. SPM analysis revealed that WIN, but not CP or M-AEA, stimulated [35S]GTPgS binding in regions that partially overlapped with the expression of CB1Rs. We then examined the role of the regulatory protein Beta-arrestin-2 (βarr2) in CB1R adaptations to chronic THC treatment. Deletion of βarr2 reduced CB1R desensitization/downregulation in the cerebellum, caudal periaqueductal gray (PAG), and spinal cord. However in hippocampus, amygdala and rostral PAG, similar desensitization was present in both genotypes. Interestingly, enhanced desensitization was found in the hypothalamus and cortex in βarr2 KO animals. Intra-regional differences in the magnitude of desensitization were noted in the caudal hippocampus, where βarr2 KO animals exhibited greater desensitization compared to WT. Regional differences in βarr2-mediated CB1R adaptation were associated with differential effects on tolerance, where THC-mediated antinociception, but not catalepsy or hypothermia, was attenuated in βarr2 KO mice. Overall, studies using SPM revealed intra- and inter-regional specificity in the function and regulation of CB1Rs and underscores an advantage of using a whole-brain unbiased approach. Understanding the regulation of CB1R signaling within different anatomical contexts represents an important fundamental prerequisite in the therapeutic exploitation of the cannabinoid system.

Les pertes de la fonction et de la masse des cellules beta pancréatiques jouent un rôle central dans le diabète de type 2 (DT2). Les beta-arrestines 1 et 2 (ARRB1 et ARRB2), sont impliquées dans la sécrétion et/ou la survie des cellules beta pancréatiques.Dans une première étude, afin de caractériser précisément la fonction d’ARRB1 dans les cellules beta pancréatiques, nous avons eu pour objectif de générer des souris invalidées spécifiquement dans ces cellules en utilisant le système Cre/lox sous le contrôle du promoteur Ins1. Des études avaient été publiées à partir des deux lignées Ins1Cre-/+ et Arrb1f/f. Nous avons généré et travaillé sur les souris Arrb1f/f :Ins1Cre-/+. Le phénotype des souris Arrb1f/f :Ins1Cre-/+ était faible et surtout non reproductible comparé aux souris Arrb1f/f :Ins1Cre-/- utilisées comme témoins. Le faible niveau d’expression d'Arrb1 dans les cellules beta et le manque d'anticorps spécifique pour l'immunocytochimie ont rendu difficile la vérification de l'absence d'expression de ARRB1 dans ces cellules. Après séquençage du gène modifié Arrb1 des souris “floxées“, nous avons pu montrer que l'insertion du premier site loxP avait induit un décalage du cadre de lecture introduisant un codon stop et, par conséquent, la non-expression du gène Arrb1. Étant donné que les souris Arrb1 “floxées“ utilisées comme témoins étaient déjà knockout (KO), le projet utilisant ces souris a dû être arrêté.Notre équipe a rapporté l'implication d'ARRB2 dans la régulation de la masse des cellules bêta pancréatique, mais son rôle dans la signalisation du récepteur du Glucagon-Like Peptide-1 (GLP-1R), une cible thérapeutique majeure du DT2, n'avait pas encore été exploré.Nous avons montré, dans une deuxième étude, une meilleure tolérance orale au glucose ainsi qu’une augmentation de la sécrétion d’insuline chez les souris Arrb2 KO par rapport aux souris témoins sur les îlots en présence des concentrations physiologiques circulantes de GLP-1. Ceci est corrélé à une production d’AMPc et un recrutement de la PKA plus élevés dans les cellules beta Arrb2 KO. A l’inverse, l’activation des kinases ERK1/2 est diminuée indiquant un recrutement majeur des ERK1/2 par ARRB2 au GLP-1R. En parallèle, j’ai montré que les taux de ARRB1 et ARRB2 des îlots pancréatiques sont altérés par des conditions diabétogènes et diabétiques. Mes résultats démontrent clairement un rôle critique de ARRB2 dans la signalisation du GLP-1R. Un défaut d’expression de la protéine pourrait participer au déficit des mécanismes de compensation de la masse fonctionnelle des cellules beta conduisant au DT2. / The loss of function and mass of pancreatic beta-cells play a central role in type 2 diabetes (T2D). Beta-arrestin 1 and 2 (ARRB1 and ARRB2) are involved in insulin secretion and/or beta-cell survival. In a first study, in order to characterize the role of ARRB1 in beta-cells, we aimed to invalidate the Arrb1 gene specifically in these cells using the Cre/lox system under the control of the Ins1 promoter. Studies had been published with both Ins1Cre-/+ and Arrb1f/f lines. We generated Arrb1f/f:Ins1Cre-/+ mice. The phenotype of Arrb1f/f :Ins1Cre-/+ mice was weak with a lack of reproducibility compared to Arrb1f/f :Ins1Cre-/- mice used as controls. The low expression level of Arrb1 in beta-cells and the lack of specific antibody for immunocytochemistry made it difficult to verify the absence of expression of ARRB1 in these cells. After sequencing the modified Arrb1 gene of the “floxed” mice, we observed that the insertion of the first loxP site induced a shift in the reading frame introducing a stop codon and, consequently, the non-expression of the Arrb1 gene. Since the “floxed“ Arrb1 mice used as controls were already knockout (KO), the project using these mice was stopped.Our team has reported the involvement of ARRB2 in the regulation of beta-cell mass, but its role in Glucagon-Like Peptide-1 (GLP-1) receptor signaling, a major therapeutic target for T2D, remained to be explored. In a second study, we showed a better glucose tolerance and an increase in insulin secretion from isolated islets in Arrb2KO compared to control mice in the presence of physiological circulating concentrations of GLP-1. This was correlated with higher cAMP production and PKA activation in Arrb2KO beta-cells. By contrast, the activation of ERK1/2 kinases was decreased indicating a major recruitment of ERK1/2 by ARRB2 to GLP-1R. In parallel, we showed that the expression levels of ARRB1 and ARRB2 in pancreatic islets were altered in diabetogenic and diabetic conditions. My results clearly demonstrate a critical role of ARRB2 in GLP-1R singaling which could impact the function, maintenance and plasticity of beta-cell mass in response to GLP-1. A lack of expression of ARRB2 could participate in the deficit of compensatory mechanisms of the functional beta-cell mass leading to T2D.

The following thesis research was undertaken to gain a better understanding of the mechanisms that regulate the cellular trafficking and signaling of the endothelial differentiation gene (EDG) family of G-protein coupled receptors, LPA1, LPA2, and LPA3. This thesis will specifically focus on the regulation of the trafficking of the LPA1 Lysophosphatidic acid receptor, which is the most widely expressed and has been shown to be a major regulator of migration of cells expressing it. The initial studies undertaken in this project were aimed at understanding the endocytic pathway followed by the LPA1 receptor. Lysophosphatidic acid (LPA), an abundant serum phospholipid, stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA1, LPA2 and LPA3. In the first part of the project we show that in addition to promoting LPA1 signaling, membrane cholesterol is essential for the association of LPA1 with β-arrestin, which leads to signal attenuation and clathrin dependent endocytosis of LPA1. The second phase of the project was aimed at elucidating the different structural motifs required for the trafficking and signaling of the LPA1 receptor and helping us gain a more mechanistic view of the processes involved in its regulation. In the second part of the project we show that agonist-independent internalization of the LPA1 receptor is clathrin adaptor, AP-2 dependent and PKC-dependent and that it requires a distal dileucine motif, whereas agonist-dependent internalization of the LPA1 receptor is β-arrestin and clathrin-dependent and requires a cluster of serine residues in the tail region, which is upstream of the dileucine motif. These studies collectively vastly enhance our understanding of mechanisms that regulate LPA1 trafficking and signaling. These studies can also be applied to other G-protein coupled receptors making the task easier for other scientists to understand this vast family of receptors.

No description available.

No description available.

<p>The following thesis will focus on understanding the downstream behavioral effects of δORmediated β-arrestinsignaling. δORagonists have been implicated as effective targets for a variety of diseases, however detrimental side effects of opioid-targeting agonists limit their clinical use. δORagonists specifically can induce seizures, however the underlying mechanism contributing to this  behavior  is  unknown.  We  review  this  phenomenon  in  more  detail,  highlighting  current agonists known to induce seizures and potential circuits and pathways involved. Our work suggests β-arrestinsignaling  is  involved,  specifically β-arrestin2  mediated  signaling  may  be  largely contributing  to δORagonist-induced  seizure  behavior.  As  it  is  possible  the β-arrestinisoforms have unique roles in seizure behavior, we also analyzed methods in which to provoke β-arrestinisoform bias of δORtargeting compounds. Though the full mechanism relating δORagonists with seizures remains unknown, our work provides foundational detail of this behavior, implicating the importance of β-arrestinisoform signaling through δOR; allowing for future studies to full define this seizure pathway and develop δORsafer agonists.  </p>