RESUMO
Arginine vasopressin (AVP) increases the water permeability of renal collecting duct principal cells by inducing the fusion of vesicles containing the water channel aquaporin-2 (AQP2) with the plasma membrane (AQP2 shuttle). This event is initiated by activation of vasopressin V2 receptors, followed by an elevation of cAMP and the activation of protein kinase A (PKA). The tethering of PKA to subcellular compartments by protein kinase A anchoring proteins (AKAPs) is a prerequisite for the AQP2 shuttle. During the search for AKAP(s) involved in the shuttle, a new splice variant of AKAP18, AKAP18delta, was identified. AKAP18delta functions as an AKAP in vitro and in vivo. In the kidney, it is mainly expressed in principal cells of the inner medullary collecting duct, closely resembling the distribution of AQP2. It is present in both the soluble and particulate fractions derived from renal inner medullary tissue. Within the particulate fraction, AKAP18delta was identified on the same intracellular vesicles as AQP2 and PKA. AVP not only recruited AQP2, but also AKAP18delta to the plasma membrane. The elevation of cAMP caused the dissociation of AKAP18delta and PKA. The data suggest that AKAP18delta is involved in the AQP2 shuttle.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Aquaporinas/química , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Rim/citologia , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Vasopressinas/química , Proteínas de Ancoragem à Quinase A , Animais , Aquaporina 2 , Arginina Vasopressina/química , Northern Blotting , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Clonagem Molecular , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Biblioteca Gênica , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Rim/metabolismo , Medula Renal/metabolismo , Cinética , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Transporte Proteico , RNA/química , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIbeta. Merlin and RIbeta demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.