G PROTEIN COUPLED RECEPTORS (GPCRS) G PROTEIN COMPLEX

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sprang and Sunahara have produced a fusion protein of beta2 adrenergic receptor (beta2AR) linked by a his6 sequence, to Galphas. This fusion protein, in the presence of Gbetagamma is able to bind specifically to beta2AR agonists and supports steady state GTP hydrolysis by the Galphas domain. The complex of beta2AR-GalphasGbeta1gamma2 can be expressed from baculovirus vectors in insect cells and purified to homogeneity in milligram quantities. Membrane extraction with dodecyl maltoside yields homogeneous micelles containing beta2AR-GalphasGbeta1gamma2. Both monomeric (~145 kDa) and dimeric complexes have been obtained. Sources of heterogeneity may derive from conformational variation and variable phosphorylation. By stimulating cAMP production and activation of Ca2+, beta2AR regulates cardiac and skeletal muscle, vascular pressure and glucose metabolism. GPCRs in general are essential regulators of neuro-endocrine, immune and sensory phenomena and are consequently major drug targets for a broad range of disorders. The mechanism of GPCR function is not understood at the molecular level. Single particle imaging of vitreous ice-embedded and negative stained beta2AR-GalphasGbeta1gamma2 particles have been undertaken in collaboration with NCMI staff. Analysis of images of negatively stained particles has demonstrated the feasibility of obtaining distinct class averages, enabling calculation of a 3-D reconstruction. These data demonstrate that beta2AR-GalphasGbeta1gamma2 particles are uniform in size, asymmetric in shape, but suffer from heterogeneity. The source of heterogeneity is under investigation, and monoclonal antibodies raised against native complexes are being developed as adjuvants to improve the stability and increase the size and asymmetry of the complexes for improved EM analysis. We seek to obtain the 3-D structure of the beta2AR-GalphasGbeta1gamma2 complex using both cryo-EM single particle imaging and x-ray crystallography, in functionally relevant conformational states. This effort is part of a long-standing research program to elucidate the mechanism of G-protein signaling.