RESUMEN
The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D3 receptor (D3R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D3R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gßγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gßγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gßγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D3R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D3R, which can be extended to the crosstalk between other RTKs and GPCRs.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores de Dopamina D3/metabolismo , Endocitosis , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Lisosomas/metabolismo , Fosforilación , Proteolisis , Proto-Oncogenes Mas , Receptores de Dopamina D3/química , Transducción de Señal/efectos de los fármacosRESUMEN
GTP binding proteins are classified into two families: heterotrimeric large G proteins which are composed of three subunits, and one subunit of small G proteins. Roles of small G proteins in the intracellular trafficking of G protein-coupled receptors (GPCRs) were studied. Among various small G proteins tested, GTP-bound form (G23V) of RalA inhibited the internalization of dopamine D2 receptor independently of the previously reported downstream effectors of RalA, such as Ral-binding protein 1 and PLD. With high affinity for GRK2, active RalA inhibited the GPCR endocytosis by sequestering the GRK2 from receptors. When it was tested for several GPCRs including an endogenous GPCR, lysophosphatidic acid receptor 1, agonist-induced conversion of GTP-bound to GDP-bound RalA, which presumably releases the sequestered GRK2, was observed selectively with the GPCRs which have tendency to undergo endocytosis. Conversion of RalA from active to inactive state occurred by translocation of RGL, a guanine nucleotide exchange factor, from the plasma membrane to cytosol as a complex with Gßγ. These results suggest that agonist-induced Gßγ-mediated conversion of RalA from the GTP-bound form to the GDP-bound form could be a mechanism to facilitate agonist-induced internalization of GPCRs.
Asunto(s)
Endocitosis/fisiología , Receptores de Dopamina D2/agonistas , Receptores del Ácido Lisofosfatídico/agonistas , Proteínas de Unión al GTP ral/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HEK293 , Humanos , Transporte de Proteínas/fisiología , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Proteínas de Unión al GTP ral/genéticaRESUMEN
Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the ß2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of ß2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.
Asunto(s)
Caveolas/metabolismo , Clatrina/metabolismo , Endocitosis/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Sulfonamidas/administración & dosificación , Tiazolidinas/administración & dosificación , Animales , Células COS , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/antagonistas & inhibidoresRESUMEN
Numerous G protein-coupled receptors (GPCRs) are glycosylated at extracellular regions. The regulatory roles of glycosylation on receptor function vary across receptor types. In this study, we used the dopamine D2and D3receptors as an experimental model to understand the underlying principles governing the functional roles of glycosylation. We used the pharmacological inhibitor, tunicamycin, to inhibit glycosylation, generated chimeric D2and D3receptors by swapping their respective N-termini, and produced the glycosylation site mutant D2and D3receptors to study the roles of glycosylation on receptor functions, including cell surface expression, signaling, and internalization through specific microdomains. Our results demonstrate that glycosylation on the N-terminus of the D3 receptor is involved in the development of desensitization and proper cell surface expression. In addition, glycosylation on the N-terminus mediates the internalization of D2and D3receptors within the caveolae and clathrin-coated pit microdomains of the plasma membrane, respectively, by regulating receptor interactions with caveolin-1 and clathrin. In conclusion, this study shows for the first time that glycosylation on the N-terminus of GPCRs is involved in endocytic pathway selection through specific microdomains. These data suggest that changes in the cellular environment that influence posttranslational modification could be an important determinant of intracellular GPCR trafficking.
Asunto(s)
Microdominios de Membrana/química , Receptores de Dopamina D2/química , Receptores de Dopamina D3/química , Secuencia de Aminoácidos , Endocitosis , Glicosilación , Células HEK293 , Humanos , Datos de Secuencia Molecular , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Tunicamicina/farmacologíaRESUMEN
A series of 4-benzylpiperidine carboxamides were designed and synthesized, and tested for their dual (serotonin and norepinephrine) reuptake inhibition. The synthesis of 4-benzylpiperidine carboxamides involved two main steps: amidation and substitution. Derivatives with 3 carbon linker displayed better activity than with 2 carbon linker. 4-Biphenyl- and 2-naphthyl-substituted derivatives 7e and 7j showed greater dual reuptake inhibition than standard drug venlafaxine HCl.
Asunto(s)
Amidas/química , Diseño de Fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/síntesis química , Inhibidores de Captación de Serotonina y Norepinefrina/síntesis química , Amidas/síntesis química , Amidas/metabolismo , Células HEK293 , Humanos , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/química , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Piperidinas/química , Unión Proteica , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/química , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo , Inhibidores de Captación de Serotonina y Norepinefrina/química , Inhibidores de Captación de Serotonina y Norepinefrina/metabolismo , Relación Estructura-ActividadRESUMEN
GPCR kinase 2 (GRK2)/ß-arrestins and protein kinase A (PKA)/protein kinase C (PKC) mediate homologous and heterologous regulations of GPCRs, respectively. Conventional protein kinase C enzymes (PKCs), as exemplified by PKCßII, selectively inhibit internalization of dopamine D2 receptor and ß2 adrenoceptor in a ß-arrestin- but not GRK2-dependent manner. PKCßII interacts with ß-arrestin2 upon autophosphorylation at T250, and inhibits the receptor internalization by decreasing the ubiquitination of ß-arrestin2. PKCßII interferes with the interaction between ß-arrestin2 and MDM2 in the cytosol, resulting in the redistribution of MDM2 to the nucleus. Subsequently, deubiquitination of ß-arrestin2 and inhibition of agonist-induced receptor internalization follow. Thus, our study suggests that the extent of ß-arrestin ubiquitination and the autophosphorylation status of PKCs determine PKCßII-mediated inhibition of homologous regulatory processes of GPCRs.