RESUMEN
Ciliary beating in the airway epithelium plays an important role in preventing infection by eliminating small particles and pathogens. Stimulation of ß2 adrenergic receptor (ß2AR) increases [cAMP]i levels and strongly activates this ciliary beating. ß2AR is localized to the apical membrane of the airways by indirectly binding to ezrin, an actin-binding protein. Ezrin takes active phosphorylated and inactive dephosphorylated states at Thr-567. Previously we showed that procaterol-stimulated ciliary beating was impaired in the ezrin-knockdown mice. In this study, we examined the roles of ezrin and its phosphorylation in regulating ciliary beating by using NSC305787, an ezrin inhibitor, in normal human airway epithelial cells (NHBE). We found that NSC305787 inhibits the phosphorylation of ezrin with an IC50 of 50 µM in NHBE. Treatment with NSC305787 for 4 h or more decreased the expression of ß2AR in the cell membrane and induced vesicle- or dot-like expression of ezrin and ß2AR inside the cell. As a result, inhibition of ezrin phosphorylation by NSC305787 attenuated the effect of procaterol-induced activation of ciliary beating in both frequency and distance indices.
Asunto(s)
Adamantano/análogos & derivados , Cilios , Proteínas del Citoesqueleto , Procaterol , Quinolinas , Ratones , Humanos , Animales , Cilios/metabolismo , Procaterol/farmacología , Procaterol/metabolismo , FosforilaciónRESUMEN
Mucociliary clearance, which is conducted by beating cilia cooperating with the surface mucous layer, is a major host defense mechanism of the airway epithelium. Ezrin, a crosslinker between membrane proteins and the actin cytoskeleton, is located in microvilli and around the basal bodies in airway ciliary cells. It is also likely that ezrin plays an important role in apical localization of ß2 adrenergic receptor (ß2AR) in airway ciliary cells. Here, we studied the physiological roles of ezrin by using trachea and airway epithelial cells prepared from ezrin-knockdown (Vil2kd/kd) mice. The trachea and airway ciliary cells of Vil2kd/kd mice presented a normal morphology and basal body orientation, suggesting that ezrin is not directly involved in development and planar cell polarity of cilia. Procaterol stimulates ciliary beating (frequency and amplitude) via ß2AR in the airway ciliary cells. In the Vil2kd/kd mice, airway ciliary beating stimulated with procaterol was partly inhibited due to the impairment of cell surface expression of ß2AR. These results suggest that ezrin regulates the beating of airway ciliary cells by promoting the apical surface localization of ß2AR. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Cilios , Procaterol , Animales , Cilios/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Humanos , Ratones , Procaterol/metabolismo , Procaterol/farmacología , Tráquea/metabolismoRESUMEN
Manipulation of adrenergic signaling has been shown experimentally and clinically to affect hair follicle growth. In this study, we provide direct evidence that canonical cAMP/CRE-binding protein signaling through adrenergic receptors can regulate hair follicle stem cell (HFSC) activation and hair cycle. We found that CRE-binding protein activation is regulated through the hair cycle and coincides with HFSC activation. Both isoproterenol and procaterol, agonists of adrenergic receptors, show the capacity to activate the hair cycle in mice. Furthermore, deletion of ADRB2 receptor, which is thought to mediate sympathetic nervous system regulation of HFSCs, was sufficient to block HFSC activation. Downstream, stimulation of adenylyl cyclase with forskolin or inhibition of phosphodiesterase to increase cAMP accumulation or direct application of cAMP was each sufficient to promote HFSC activation and accelerate initiation of hair cycle. Genetic induction of a Designer Receptors Exclusively Activated by Designer Drug allele showed that G-protein coupled receptor/GαS stimulation, specifically in HFSCs, promoted the activation of the hair cycle. Finally, we provide evidence that G-protein coupled receptor/CRE-binding protein signaling can potentially act on HFSCs by promoting glycolytic metabolism, which was previously shown to stimulate HFSC activation. Together, these data provide mechanistic insights into the role of sympathetic innervation on HFSC function.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , AMP Cíclico/metabolismo , Folículo Piloso/fisiología , Cabello/fisiología , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/fisiología , Animales , Diferenciación Celular , Glucólisis , Cabello/patología , Isoproterenol/metabolismo , Queratina-15/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procaterol/metabolismo , Receptores Adrenérgicos beta 2/genética , Transducción de Señal , Sistema Nervioso SimpáticoRESUMEN
G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human ß(2) adrenergic receptor (ß(2)AR) as a guide, we designed a ß(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent ß(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound ß(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 µs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Cristalización , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Agonismo Inverso de Drogas , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Procaterol/química , Procaterol/metabolismo , Propanolaminas/química , Propanolaminas/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
As indicated in the Global Initiative for Asthma guidelines, short-acting ß2-adrenoreceptor agonists (SABAs) are important relievers in asthma exacerbation. Interferon γ-inducible protein (IP)-10/CXCL 10 is a T-helper type 1 (Th1) cell-related chemokine which is important in the recruitment of Th1 cells involved in host immune defense against intracellular pathogens such as viral infection. Regulated on activation, normal T expressed and secreted (RANTES)/CCL 5 is a chemokine which plays a role in attractant of eosinophils, mast cells, and basophils toward the site of allergic inflammation. Bronchial epithelial cells are first-line barriers against pathogen invasion. However, whether SABAs have regulatory effects on the expression of IP-10 and RANTES in bronchial epithelial cells is unknown. BEAS-2B cells, the human bronchial epithelial cell lines, were pretreated with procaterol (one of the SABAs) or dibutyryl-cAMP (a cyclic AMP analog) at different doses for 1 h and then stimulated with poly I:C (10 µg/mL). Supernatants were collected 12 and 24 h after poly I:C stimulation to determine the concentrations of IP-10 and RANTES by ELISA. In some cases, the cells were pretreated with selective ß2-adrenoreceptor antagonist, ICI-118551, 30 min before procaterol treatment. To investigate the intracellular signaling, the cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitors and a NF-κB inhibitor 30 min before procaterol treatment. Western blot was also used to explore the intracellular signaling. Procaterol significantly suppressed poly I:C-induced IP-10 and RANTES in BEAS-2B cells in a dose-dependent manner. ICI-118551, a selective ß2-adrenoreceptor antagonist, could significantly reverse the suppressive effects. Dibutyryl-cAMP could confer the similar effects of procaterol on poly I:C-induced IP-10 and RANTES expression. Data of Western blot revealed that poly I:C-induced p-ERK, p-JNK, and pp38 expression, but not pp65, were suppressed by procaterol. SABAs could suppress poly I:C-induced IP-10 and RANTES expression in bronchial epithelial cells, at least in part, via ß2-adrenoreceptor-cAMP and MAPK-ERK, JNK, and p38 pathways.
Asunto(s)
Bronquios/metabolismo , Quimiocina CCL5/biosíntesis , Quimiocina CXCL10/biosíntesis , Células Epiteliales/metabolismo , Procaterol/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Western Blotting , Bronquios/efectos de los fármacos , Bronquios/inmunología , Bucladesina/farmacología , Línea Celular , Quimiocina CCL5/genética , Quimiocina CXCL10/genética , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/inmunología , FN-kappa B/antagonistas & inhibidores , Poli I-C/farmacología , Procaterol/metabolismo , Propanolaminas/farmacología , Transducción de Señal/efectos de los fármacos , eIF-2 Quinasa/biosíntesisRESUMEN
1. Using the cannula inserting method, vasodilator responses to beta-adrenoceptor agonists (isoprenaline, denopamine and procaterol) were investigated in isolated and perfused rat common carotid arteries. 2. Each beta-adrenoceptor agonist induced a vasodilation in preparations preconstricted by phenylephrine in a dose-related manner. The potencies were in the order of isoprenaline > procaterol >> denopamine. 3. Denopamine-induced dilations were significantly inhibited by 1 nmol betaxolol (a selective beta1-adrenoceptor antagonist), but it was not influenced by 1 nmol ICI 118,551 (a selective beta2-adrenoceptor antagonist). On the other hand, procaterol-induced vasodilations were significantly inhibited by 1 nmol ICI 118,551 but not modified by 10 nmol betaxolol. 4. ACh-induced vasodilations disappeared after intraluminal saponin injection to remove endothelium, but procaterol- and denopamine-induced dilations were not modified by removal of the endothelium. 5. Pretreatment with L-NG-nitroarginine methyl ester (L-NAME) readily inhibited ACh-induced vasodilations. However, neither procaterol- or denopamine-induced vasodilation was modified by L-NAME treatment. 6. From these results, it is concluded that in the rat common carotid arteries (1) there are abundant beta2- and a few beta1-adrenoceptors, and (2) there is no participation of the endothelium-dependent mechanism in beta-adrenoceptor mediated vasodilations.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 1 , Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacología , Arterias Carótidas/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Etanolaminas/farmacología , Isoproterenol/farmacología , Procaterol/metabolismo , Procaterol/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Betaxolol/farmacología , Relación Dosis-Respuesta a Droga , Etanolaminas/metabolismo , Femenino , Masculino , NG-Nitroarginina Metil Éster/farmacología , Perfusión , Propanolaminas/farmacología , Ratas , Ratas Wistar , Receptores Colinérgicos/metabolismo , Saponinas/farmacología , VasodilataciónRESUMEN
We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.