RESUMEN
Truncations at the carboxyl termini of G protein-coupled receptors result in defective receptor biogenesis and comprise a number of inherited disorders. In order to evaluate the structural role of the C-terminus in G protein-coupled receptor biogenesis, we generated a series of deletion and substitution mutations in the dopamine D1 receptor and visualized receptor subcellular localization by fusion to a green fluorescent protein. Alanine substitutions of several hydrophobic residues within the proximal C-terminus resulted in receptor transport arrest in the ER. Agonist binding and coupling to adenylyl cyclase was also abolished. In contrast, substitutions conserving C-terminal hydrophobicity produced normal cell surface receptor expression, binding, and stimulatory function. A mechanism for the role of the C-terminus in D1 receptor transport was investigated by searching for candidate protein interactions. The D1 receptor was found to co-precipitate and associate in vitro directly with the gamma-subunit of the COPI coatomer complex. In vitro pull-down assays confirmed that only the D1 C-terminus is required for COPI association, and that identical mutations causing disruption of receptor transport to the cell surface also disrupted binding to COPI. Furthermore, conservative mutations in the D1 C-terminus restored COPI association just as they restored cell surface transport. These results suggest that association between the coatomer complex and hydrophobic residues within the proximal C-terminus of the D1 receptor may serve an important role in receptor transport.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteína Coatómero , Células Eucariotas/metabolismo , Proteínas de Unión al GTP/metabolismo , Membranas Intracelulares/metabolismo , Transporte de Proteínas/fisiología , Receptores de Dopamina D1/biosíntesis , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos/genética , Animales , Sitios de Unión/genética , Membrana Celular/ultraestructura , Células Cultivadas , Células Eucariotas/ultraestructura , Proteínas Fluorescentes Verdes , Humanos , Indicadores y Reactivos , Membranas Intracelulares/ultraestructura , Proteínas Luminiscentes , Mutagénesis/genética , Mutación/genética , Unión Proteica/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Receptores de Superficie Celular/metabolismo , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/genéticaRESUMEN
Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Contráctiles/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores de Dopamina D2/metabolismo , Inhibidores de Adenilato Ciclasa , Filaminas , Humanos , Ensayo de Unión Radioligante , Receptores de Dopamina D3 , Células Tumorales Cultivadas , Técnicas del Sistema de Dos HíbridosRESUMEN
The RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway provides numerous opportunities for targeted oncology therapeutics. In particular, the MEK enzyme is attractive due to high selectivity for its target ERK and the central role that activated ERK plays in driving cell proliferation. The structural, pharmacologic, and pharmacokinetic properties of RDEA119/BAY 869766, an allosteric MEK inhibitor, are presented. RDEA119/BAY 869766 is selectively bound directly to an allosteric pocket in the MEK1/2 enzymes. This compound is highly efficacious at inhibiting cell proliferation in several tumor cell lines in vitro. In vivo, RDEA119/BAY 869766 exhibits potent activity in xenograft models of melanoma, colon, and epidermal carcinoma. RDEA119/BAY 869766 exhibits complete suppression of ERK phosphorylation at fully efficacious doses in mice. RDEA119/BAY 869766 shows a tissue selectivity that reduces its potential for central nervous system-related side effects. Using pharmacokinetic and pharmacodynamic data, we show that maintaining adequate MEK inhibition throughout the dosing interval is likely more important than achieving high peak levels because greater efficacy was achieved with more frequent but lower dosing. Based on its longer half-life in humans than in mice, RDEA119/BAY 869766 has the potential for use as a once- or twice-daily oral treatment for cancer. RDEA119/BAY 869766, an exquisitely selective, orally available MEK inhibitor, has been selected for clinical development because of its potency and favorable pharmacokinetic profile.