RESUMO
The beta-adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2), its negative regulator phospholamban (PLN), the A-kinase anchoring protein AKAP18delta and PKA. We show that AKAP18delta acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca(2+) re-uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca(2+) re-uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Complexos Multiproteicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Retículo Sarcoplasmático/ultraestrutura , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Alinhamento de SequênciaRESUMO
Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.
Assuntos
Aquaporina 2/metabolismo , Proteínas de Transporte/fisiologia , Rim/metabolismo , Proteínas de Membrana/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Animais , Aquaporina 2/biossíntese , Proteínas de Transporte/biossíntese , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Rim/citologia , Proteínas de Membrana/biossíntese , Cadeias Pesadas de Miosina/biossíntese , Miosina Tipo V/biossíntese , Miosinas/biossíntese , Ligação Proteica , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas rab de Ligação ao GTPRESUMO
The cAMP/protein kinase A (PKA)-dependent insertion of water channel aquaporin-2 (AQP2)-bearing vesicles into the plasma membrane in renal collecting duct principal cells (AQP2 shuttle) constitutes the molecular basis of arginine vasopressin (AVP)-regulated water reabsorption. cAMP/PKA signaling systems are compartmentalized by A kinase anchoring proteins (AKAP) that tether PKA to subcellular sites and by phosphodiesterases (PDE) that terminate PKA signaling through hydrolysis of localized cAMP. In primary cultured principal cells, AVP causes focal activation of PKA. PKA and cAMP-specific phosphodiesterase-4D (PDE4D) are located on AQP2-bearing vesicles. The selective PDE4 inhibitor rolipram increases AKAP-tethered PKA activity on AQP2-bearing vesicles and enhances the AQP2 shuttle and thereby the osmotic water permeability. AKAP18delta, which is located on AQP2-bearing vesicles, directly interacts with PDE4D and PKA. In response to AVP, PDE4D and AQP2 translocate to the plasma membrane. Here PDE4D is activated through PKA phosphorylation and reduces the osmotic water permeability. Taken together, a novel, compartmentalized, and physiologically relevant cAMP-dependent signal transduction module on AQP2-bearing vesicles, comprising anchored PDE4D, AKAP18delta, and PKA, has been identified.
