RESUMEN
The ß2 adrenergic receptor (ß2AR) signals through both Gs and Gi in cardiac myocytes, and the Gi pathway counteracts the Gs pathway. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study ß2AR-Gi interactions. Here we investigate the role of phospholipid composition on Gs and Gi coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the ß2AR, we find that they impair coupling to Gi3 and facilitate coupling to Gs. Positively charged Ca2+ and Mg2+, known to interact with the negative charge on phospholipids, facilitates Gi3 coupling. Mutational analysis suggests that Ca2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of Gi3. Taken together, our observations suggest that local membrane charge modulates the interaction between ß2AR and competing G protein subtypes.
Asunto(s)
Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Lípidos de la Membrana/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Secuencias de Aminoácidos , Animales , Cationes/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/aislamiento & purificación , Lípidos de la Membrana/química , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Electricidad EstáticaRESUMEN
Transient or partial formation of complexes between biomacromolecules is a general mechanism used to control cellular functions. Several of these complexes escape structure determination by crystallographic means. We developed a new approach for determining the structure of protein dimers in the native environment (e.g., in the membrane) with high resolution in cases where the structure of the two monomers is known. The approach is based on measurements of distance distributions between spin labels in the range between 2 and 6 nanometers by a pulsed electron paramagnetic resonance technique and explicit modeling of spin label conformations. By applying this method to the membrane protein homodimer of the Na(+)/H(+) antiporter NhaA of Escherichia coli, the structure of the presumably physiological dimer was determined. It reveals two points of contact between the two monomers, with one of them confirming results of earlier cross-linking experiments.
Asunto(s)
Intercambiadores de Sodio-Hidrógeno/química , Alelos , Sitios de Unión , Microscopía por Crioelectrón , Cisteína/química , Bases de Datos de Proteínas , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Modelos Estadísticos , Conformación Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Amino acid transport is a ubiquitous phenomenon and serves a variety of functions in prokaryotes, including supply of carbon and nitrogen for catabolic and anabolic processes, pH homeostasis, osmoprotection, virulence, detoxification, signal transduction and generation of electrochemical ion gradients. Many of the participating proteins have eukaryotic relatives and are successfully used as model systems for exploration of transporter structure and function. Distribution, physiological roles, functional properties, and structure-function relationships of prokaryotic alpha-amino acid transporters are discussed.
