Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast.

We describe a procedure for isolating agonists for mammalian G protein-coupled receptors of unknown function. Human formyl peptide receptor like-1 (FPRL-1) receptor, originally identified as an orphan G protein-coupled receptor related to the formyl peptide receptor (FPR1), was expressed in Saccharomyces cells designed to couple receptor activation to histidine prototrophy. Selection for histidine prototrophs among transformants obtained with a plasmid-based library encoding random peptides identified six different agonists, each of whose production yielded autocrine stimulation of the receptor expressed in yeast. A synthetic version of each peptide promoted activation of FPRL-1 expressed in human embryonic kidney (HEK293) cells, and five of the peptides exhibited significant selectivity for activation of FPRL-1 relative to FPR1. One selective peptide was tested and found to mobilize calcium in isolated human neutrophils. This demonstrates that stimulation of FPRL-1 results in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response. This autocrine selection protocol may be a generally applicable method for providing pharmacological tools to evaluate the physiological roles of the growing number of mammalian orphan G protein-coupled receptors.

Corpus callosum involvement as a prognostic factor for patients with high-grade astrocytoma.

PURPOSE: To evaluate corpus callosum involvement as a prognostic factor for patients with high-grade astrocytoma. METHODS: From 1986 through 1994, 141 adult patients with Karnofsky performance status (KPS) > or = 40 underwent primary treatment for anaplastic astrocytoma (AA) or glioblastoma multiforme (GBM) at the University of Washington Medical Center. Preoperative magnetic resonance imaging and/or computed tomography to assess corpus callosum involvement was available for 105 of these patients. Corpus callosum involvement was evaluated as a prognostic factor for survival using recursive partitioning analysis and multivariate analysis with a Cox proportional hazards model. RESULTS: For the 105 patients evaluable for corpus callosum involvement, the median and 2-year survival were 59 weeks and 28%, respectively. On multivariate analysis, the only independent prognostic factors were KPS (p = 0.0001) and histology (p = 0.042). On recursive partitioning analysis, the first significant split occurred at KPS < 70 vs. KPS > or = 70. Patients with KPS > or = 70 were split by age (< 50 years vs. > or = 50 years), with those younger than 50 years further split by absence or presence of corpus callosum involvement. Among patients with KPS > or = 70 and age < 50 years, median survival was 57 weeks if the corpus callosum was involved (35% 2-year survival) and 105 weeks if the corpus callosum was not involved (56% 2-year survival). CONCLUSION: Corpus callosum involvement based on preoperative imaging is an unfavorable prognostic factor for survival among the subgroup of young, good-performance-status patients with high-grade astrocytoma.

Coordinate regulation of estrogen receptor ß degradation by Mdm2 and CREB-binding protein in response to growth signals.

The biological actions of estrogen are mediated via estrogen receptors ERα and ERß. Yet, other cellular signaling events that also impact ER functions have an important role in breast carcinogenesis. Here, we show that activation of ErbB2/ErbB3 tyrosine kinase receptors with growth factor heregulin-ß prompts ERß degradation by the 26S proteasome, a mechanism that requires the coactivator cAMP response element-binding (CREB)-binding protein (CBP). We found that CBP promotes ERß ubiquitination and degradation through enhancement of the PI3-K/Akt pathway by heregulin-ß, an effect potentiated by a negatively charged hinge region of ERß. Activated Akt triggered the recruitment of E3 ubiquitin ligase Mdm2 to ERß, which was further stabilized by CBP, resulting in ERß poly-ubiquitination. Mutation of CBP Thr-1872 or Mdm2 Ser-186/188 Akt sites resulted in a dissociation of the ERß-CBP-Mdm2 complex and reduced ERß turnover. We found that the decrease in ERß induced by heregulin-ß was associated with reduced target gene promoter occupancy and enhanced proliferation of breast cancer cells. However, knockdown of Mdm2 restored endogenous ERß levels resulting in reduction of breast cancer cell growth. These studies identify a tripartite Akt-regulated phosphorylation mechanism that functions to hamper normal ERß activity and turnover through the concerted actions of CBP and Mdm2 in response to growth factor signaling pathways in breast cancer cells.

Gamma-band synchronous oscillations: recent evidence regarding their functional significance.

How do our brains represent distinct objects in consciousness? In order to consciously distinguish between objects, our brains somehow selectively bind together activity patterns of spatially intermingled neurons that simultaneously represent similar and dissimilar features of distinct objects. Gamma-band synchronous oscillations (GSO) of neuroelectrical activity have been hypothesized to be a mechanism used by our brains to generate and bind conscious sensations to represent distinct objects. Most experiments relating GSO to specific features of consciousness have been published only in the last several years. This brief review focuses on a wide variety experiments in which animals, including humans, discriminate between sensory stimuli and make these discriminations evident in their behavior. Performance of these tasks, in humans, is invariably accompanied by conscious awareness of both stimuli and behavior. Results of these experiments indicate that specific patterns of GSO correlate closely with specific aspects of conscious sensorimotor processing. That is, GSO appear to be closely correlated with neural generation of our most paradigmatic cognitive state: consciousness.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.