R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

<p>Abstract</p> <p>Background</p> <p>Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS) proteins of the R7 subfamily (R7-RGS). The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP), a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain.</p> <p>Results</p> <p>We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain.</p> <p>Conclusion</p> <p>A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of R7BP regulated the lipid raft targeting of endogenous or co-expressed Gβ5/R7-RGS proteins. Taken together with recent evidence that the kinetic effects of the Gβ5 complex on GPCR signaling are greatly enhanced by R7BP palmitoylation through a membrane-anchoring mechanism, our data suggest the targeting of the Gβ5/R7-RGS/R7BP complex to lipid rafts in neurons and brain, where G proteins and their effectors are concentrated, may be central to the G protein regulatory function of the complex.</p

Altered Expression of Insulin Receptor Isoforms in Breast Cancer

PURPOSE: Insulin-like growth factor (IGF) signaling through human insulin receptor isoform A (IR-A) contributes to tumorigenesis and intrinsic resistance to anti-IGF1R therapy. In the present study, we (a) developed quantitative TaqMan real time-PCR-based assays (qRT-PCR) to measure human insulin receptor isoforms with high specificity, (b) evaluated isoform expression levels in molecularly-defined breast cancer subtypes, and (c) identified the IR-A:IR-B mRNA ratio as a potential biomarker guiding patient stratification for anti-IGF therapies. EXPERIMENTAL DESIGN: mRNA expression levels of IR-A and IR-B were measured in 42 primary breast cancers and 19 matched adjacent normal tissues with TaqMan qRT-PCR assays. The results were further confirmed in 165 breast cancers. The tumor samples were profiled using whole genome microarrays and subsequently subtyped using the PAM50 breast cancer gene signature. The relationship between the IR-A:IR-B ratio and cancer subtype, as well as markers of proliferation were characterized. RESULTS: The mRNA expression levels of IR-A in the breast tumors were similar to those observed in the adjacent normal tissues, while the mRNA levels of IR-B were significantly decreased in tumors. The IR-A:IR-B ratio was significantly higher in luminal B breast cancer than in luminal A. Strong concordance between the IR-A:IR-B ratio and the composite Oncotype DX proliferation score was observed for stratifying the latter two breast cancer subtypes. CONCLUSIONS: The reduction in IR-B expression is the key to the altered IR-A:IR-B ratio observed in breast cancer. The IR-A:IR-B ratio may have biomarker utility in guiding a patient stratification strategy for an anti-IGF therapeutic

Pneumocystis Encodes a Functional S-Adenosylmethionine Synthetase Gene▿

S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6) is the enzyme that catalyzes the synthesis of AdoMet, a molecule important for all cellular organisms. We have cloned and characterized an AdoMet synthetase gene (sam1) from Pneumocystis spp. This gene was transcribed primarily as an ∼1.3-kb mRNA which encodes a protein containing 381 amino acids in P. carinii or P. murina and 382 amino acids in P. jirovecii. sam1 was also transcribed as part of an apparent polycistronic transcript of ∼5.6 kb, together with a putative chromatin remodeling protein homologous to Saccharomyces cerevisiae, CHD1. Recombinant Sam1, when expressed in Escherichia coli, showed functional enzyme activity. Immunoprecipitation and confocal immunofluorescence analysis using an antipeptide antibody showed that this enzyme is expressed in P. murina. Thus, Pneumocystis, like other organisms, can synthesize its own AdoMet and may not depend on its host for the supply of this important molecule

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-6

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>alyzed by immunoblotting for flotillin-1, as marker for DRM, and the transferrin receptor (TfR) as a non-DRM marker. B. Sucrose density gradient analysis of endogenous Gβ5, RGS7, and R7BP synaptosomal membrane proteins in mouse brain. A detergent extract of mouse brain synaptosomes was subjected to a sucrose density gradient fractionation. Equal aliquots from all eleven fractions were subjected to SDS-PAGE followed by immunoblotting for the endogenous Gβ5, RGS7, and R7BP (using antibody TRS) proteins as indicated. Immunoblots of the sucrose density gradients shown were analyzed by densitometry and the distribution of the indicated immunoreactive bands between DRM and non-DRM fractions among (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblot, with error bars representing the S.E.M

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-7

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>AGE gels and subjected to immunoblotting using anti-R7BP (N-terminal), anti-RGS7, anti-Gβ5, and actin antibodies as described in Methods. The relative mobility of the major immunoreactive bands (in kDa) is indicated on the right. B. Determination of DRM (lipid raft) and non-DRM markers along a sucrose density gradient in PC12 cells. The DRM (lipid rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation as detailed in Methods. Fractions were analyzed by immunoblotting for flotillin-1, LAT and PSD-95 as markers for DRM and the β2 subunit of Na-K ATPase as a non-DRM marker. Fractions 1–5 were enriched in the DRM marker, while fractions 6–10 carried the non-DRM marker. C. Localization of endogenous Gβ5 and RGS7 proteins in PC12 cells. PC12 cells were lysed, and subjected to a sucrose density gradient. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the endogenous Gβ5 and RGS7 proteins as indicated. D. Densitometric quantification of the distribution of endogenous Gβ5 and RGS7 proteins among DRM and non-DRM fractions in sucrose density gradients. Immunoblots of sucrose density gradients such as that shown in C were analyzed by densitometry and the distribution of the indicated immunoreactivity between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblots, with error bars representing the S.E.M. The results shown are representative of three experiments completed with similar results

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-0

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>AGE gels and subjected to immunoblotting using anti-R7BP (N-terminal), anti-RGS7, anti-Gβ5, and actin antibodies as described in Methods. The relative mobility of the major immunoreactive bands (in kDa) is indicated on the right. B. Determination of DRM (lipid raft) and non-DRM markers along a sucrose density gradient in PC12 cells. The DRM (lipid rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation as detailed in Methods. Fractions were analyzed by immunoblotting for flotillin-1, LAT and PSD-95 as markers for DRM and the β2 subunit of Na-K ATPase as a non-DRM marker. Fractions 1–5 were enriched in the DRM marker, while fractions 6–10 carried the non-DRM marker. C. Localization of endogenous Gβ5 and RGS7 proteins in PC12 cells. PC12 cells were lysed, and subjected to a sucrose density gradient. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the endogenous Gβ5 and RGS7 proteins as indicated. D. Densitometric quantification of the distribution of endogenous Gβ5 and RGS7 proteins among DRM and non-DRM fractions in sucrose density gradients. Immunoblots of sucrose density gradients such as that shown in C were analyzed by densitometry and the distribution of the indicated immunoreactivity between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblots, with error bars representing the S.E.M. The results shown are representative of three experiments completed with similar results

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-5

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>d rafts) were isolated using a sucrose density gradient prepared by ultracentrifugation. Fractions were analyzed by immunoblotting for Fyn as a marker for DRM and the α1 subunit of Na-K ATPase as a non-DRM marker. Fractions 1–5 were enriched in the DRM marker, while fractions 6–10 carried the non-DRM marker. B. Localization of transfected AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were transfected with the indicated constructs, lysed and subjected to a sucrose density gradient centrifugation. Equal aliquots from all ten fractions were subjected to SDS-PAGE followed by immunoblotting for the transfected Gβ5 and RGS7 proteins as indicated. Immunoblots of the sucrose density gradients shown were analyzed by densitometry and the distribution of the indicated immunoreactive bands between DRM and non-DRM fractions (as % of total immunoreactivity with that antibody) is shown as a histogram to the right of the corresponding immunoblot, with error bars representing the S.E.M. C. Localization of co-transfected wild type HA-R7BP, AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were co-transfected as indicted and analyzed as indicated in the legend to panel B. D. Localization of co-transfected HA-R7BP-SS, AU5-Gβ5 and RGS7 proteins in HEK-293 cells. Cells were co-transfected as indicated and analyzed as indicated in the legend to panel B. These results are representative of four total such experiments performed with similar findings

R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain-3

<p><b>Copyright information:</b></p><p>Taken from "R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain"</p><p>http://www.biomedcentral.com/1471-2091/8/18</p><p>BMC Biochemistry 2007;8():18-18.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2048962.</p><p></p>ractionated into Triton X-100 soluble and insoluble fractions. Proteins in each fraction were probed for HA-R7BP, Gβ5 and RGS7 and α-tubulin by immunoblotting with specific antibodies as shown. The data shown are representative of three experiments performed with similar results. B. The percentage of total transfected HA-R7BP or endogenous Gβ5 or RGS7 protein in the Triton X-100 soluble (Sol) or insoluble (Insol) fractions was determined by densitometry for the different transfection conditions as shown, with error bars representing the S.E.M