Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.

SOCS-1 is an inducible SH2-containing inhibitor of Jak kinases and as such can potently suppress cytokine signaling. SOCS-1 de®cient mice die within the ®rst three weeks of life from a myeloproliferative disorder driven by excessive interferon signaling. We report here that SOCS-1 inhibits proliferation signals induced by a variety of oncogenes active within the hematopoietic system. Ectopic expression of SOCS-1 abolished proliferation mediated by a constitutively active form of the KIT receptor, TEL-JAK2, and v-ABL, and reduced metastasis from BCR-ABL transformed cells. SOCS-1, however, did not interfere with v-SRC or RASV12 mediated cellular transformation. A mutant form of SOCS-1 unable to bind through its SH2 domain to tyrosine phosphorylated proteins could still inhibit KIT, but not TEL-JAK2, indicating multiple mechanisms for SOCS-1-mediated tumor suppression. We show that the steady state levels of TEL-JAK2 and to a greater extent v-ABL are diminished in the presence of SOCS-1. Lastly, we show that SOCS-1 7/7 ®broblasts are more sensitive than wild type ®broblasts to either spontaneous or oncogene-induced transformation. These data suggest that loss-of-function of SOCS-1 may collaborate with a variety of hematopoietic oncogenes to facilitate tumor progression.

No abstract

Driver mutations in KRAS occur in almost 30% of human tumors, primarily in pancreatic, colorectal and lung tumors. These mutations result in increased cell proliferation and survival predominantly mediated through the MAPK signaling pathway. MAPK signal transduction is initiated by the interaction of RAF kinase with active RAS at the plasma membrane. The precise molecular details of this process are currently unknown. The Frederick National Laboratory for Cancer Research has partnered with the Department of Energy to harness high-performance computing and experimental data to generate models and hypotheses of how KRAS engages with RAF kinase at the plasma membrane to initiate signal transduction. The initial phase of this work has focused on identifying membrane bound states of KRAS. We have used a variety of biophysical approaches (including NMR, protein foot-printing and neutron reflectivity) to investigate the structural orientation of KRAS at the membrane. In addition, large scale coarse-grained simulations of membrane bound KRAS spanning the millisecond time scale, have been completed. Three predominant membrane bound KRAS states were observed computationally: an exposed state (where switch 1 is available for RAF binding), an occluded state (where switch 1 is unavailable for RAF binding) and a transition state (where helix 5 is perpendicular to the membrane). These three states are also identified in the experimental data. Cumulatively, experimental and computational data predict KRAS exists in a dynamic equilibrium on the plasma membrane, interconverting between 3 states on the nanosecond time scale. The experimental data indicates the most populated conformation of KRAS is the transition state. Future efforts will address the significance of these three states for RAF interaction and signal transduction. This in depth understanding of RAS activation of RAF and the MAPK pathway is critically important for developing effective therapeutic interventions for cancers harboring mutant RAS. Citation Format: Andrew G. Stephen, Animesh Agarwal, Angel E. Garcia, Gnana S. Gnanakaran, Jeevapani Hettige, Christopher Neale, Timothy Travers, Harsh Bhatia, Peer-Timo Bremer, Tim Carpenter, Jim Glosli, Helgi Ingolfsson, Piyush Karande, Felice Lightstone, Tomas Oppelstrup, Liam Stanton, Shiv Sundram, Xiaohua Zhang, Debsindhu Bhowmik, Arvind Ramanathan, Christopher Stanley, Debanjan Goswami, Gulcin Gulten, Frantz Jean-Francios, Dhirendra Simanshu, Tommy Turbyville, Rebika Shrestha, Que Van, Frank McCormick, Dwight Nissley, Fred Streitz, Constance Agamasu. Identification of KRAS membrane bound states using an integrated computational and experimental approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3373.

No abstract