archives@tulane.edu / Glucocorticoid-induced negative feedback of the hypothalamic-pituitary-adrenal axis is rapidly achieved by two mechanisms: desensitization of the corticotropin releasing hormone-producing neurons to excitatory noradrenergic inputs by internalization of the α-1 receptor and suppression of excitatory synaptic input via production of an endocannabinoid retrograde messenger. Several previously undetermined signaling factors in the glucocorticoid-induced endocannabinoid release were identified here. The glucocorticoid-induced desensitization to adrenergic receptor signaling was revealed to involve α-1 receptor intracellular trafficking to the late endosome. The physiological significance and therapeutic targets of attenuation of the stress response to noradrenergic inputs was also investigated using electrophysiology and pharmacogenetics. These experiments indicate that desensitization to norepinephrine selectively attenuates the stress response to physiological over psychological stressors. These rapid interactions between glucocorticoids and adrenoreceptor trafficking and endocannabinoid synthesis represent novel glucocorticoid signaling mechanisms through G-protein coupled receptors. / 1 / Grant L. Weiss

No description available.

The study of arrestins as regulators of seven transmembrane receptor (7TMR) signaling has revealed multiple levels of complexity, initiating desensitization of G protein activity and coordination of receptor internalization via clathrin‐coated pits. Recently, β‐arrestins have also been shown to act as adaptor proteins, mediating G protein‐independent signaling as well as scaffolding of enzymes that degrade second messenger molecules. This latter function was demonstrated by β‐arrestins recruiting PDE4 phosphodiesterase to Gs‐coupled β2‐adrenergic receptors, enhancing metabolism of the second messenger cAMP. As β‐arrestins universally interact with members of the 7TMR superfamily, we sought to determine if this phenomenon of concerted desensitization might be applicable to additional receptor subtypes. We screened for β‐arrestin‐binding proteins among modulators of diacylglycerol and IP3 (second messengers downstream of Gq‐coupled 7TMRs). We observed β‐ arrestins constitutively interacted with members of the diacylglycerol kinase (DGK) family, which phosphorylate diacylglycerol to create phosphatidic acid. Furthermore, examining lipid extracts of 32P labeled cells separated by TLC, we observed that overexpression of β‐arrestin enhanced phosphatidic acid (PA) production after M1 muscarinic receptor stimulation. Conversely, depletion of β‐arrestins by RNA interference showed significantly decreased agonist‐stimulated PA accumulation. Additionally, overexpression of a β‐arrestin2 mutant that binds DGKs but not receptors served as a dominant negative for agonist‐dependent DGK activity. These results demonstrate a requirement for β‐arrestins in DGK translocation to the membrane, and specifically to activated 7TMRs, where concentrations of second messengers are at their highest. Phosphatidic acid is an effector for several enzymes, including the phosphatidylinositol 5‐kinases (PIP5K), which phosphorylate PIP to make PIP2. Thus, we hypothesized β‐arrestin‐targeted DGKs may regulate PIP5K activity. PIP5K Iα associated with β‐arrestin2 in an agonist‐dependent manner in HEK293 cells, and a β‐ arrestin2 mutant defective in receptor endocytosis (a PIP2‐dependent function) was impaired. Furthermore, knockdown of β‐arrestin2 by RNAi significantly decreased the amount of PIP5K Iα detected in receptor immunoprecipitates. In TLC assays, overexpressing both β‐arrestin2 and PIP5K Iα enhanced agonist‐stimulated PIP2 labeling, while either protein alone had no effect. These data support the concept of β‐ arrestin binding to 7TMRs and enriching local membrane concentrations of PA, which then stimulates production of PIP2, promoting receptor internalization. / Dissertation

<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

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 107-117).

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23366号 / 医博第4735号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 萩原 正敏, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Beta-arrestins have been suggested to regulate μ-, δ-, and κ-opioid receptor-mediated responses. In the present study, we examined the effects of pretreatment with β-arrestin-2 antibody on tail-flick inhibition induced by opioid receptor agonists in the mouse spinal cord. Intrathecal (i.t.) pretreatment with β-arrestin-2 antibody potentiated the antinociception induced by i.t.-administered μ-opioid receptor agonists [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) and endomorphin-1, but not endomorphin-2, the δ-opioid receptor agonist [D-Ala2]deltorphin II or the κ-opioid receptor agonist U50,488H. The present result suggests that β-arrestin-2 may tonically down-regulate a selected population of μ-opioid receptors activated by endomorphin-1 or DAMGO in the mouse spinal cord.

Physical and psychological stress can alter the immune system in both humans and animals. Stress is a known risk factor for numerous human diseases, such as infectious and autoimmune diseases, and cancer. Toll-like receptors (TLRs) play a pivotal role in the induction of innate and adaptive immune response. Our previous studies have shown that TLR4 deficiency prevents stress-induced splenocyte reduction. However, the role of TLR2 in stress-mediated lymphocyte reduction is unknown. In this study, we investigated the effects of TLR2 ligands on stress-induced lymphocyte reduction. We also defined whether the phosphoinositide 3-kinases (PI3Ks)/Akt pathway contributes to TLR2-mediated lymphocyte numbers altered by stress. Our data have shown that stimulation of TLR2 by TLR2 ligands peptidoglycan (PGN) or Pam3CSK4 (Pam3) attenuates stress-induced reduction in lymphocyte numbers. However, TLR2 ligand-induced protection from stress-induced lymphocyte reduction is lost in TLR2 deficiency in mice. Furthermore, stimulation of TLR2 by PGN induces protection from stress-induced reduction in the number of splenocytes through PI3K. Moreover, PGN dramatically increases the level of phosphorylation of Akt through a PI3K-dependent manner. Moreover, we found that stimulation of TLR2 by PGN induced protection from stress-induced reduction in splenocyte numbers is abolished in β-arrestin 2 deficient mice. In addition, PGN-induced immune protection in stress-induced changes of cytokine levels appears to require -arrestin 2, a multifunctional adaptor and signal transducer. Collectively, our study thus demonstrates that stimulation of TLR2-mediated PI3K signaling attenuates splenocyte reduction induced by stress, and that β-arrestin 2 modulates TLR2-mediated immune response following stress.

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.