Proteins in the transforming growth factor-␤ (TGF-␤) family recognize transmembrane serine/threonine kinases known as type I and type II receptors. Binding of TGF-␤ to receptors results in receptor down-regulation and signaling. Whereas previous work has focused on activities controlling TGF-␤ signaling, more recent studies have begun to address the trafficking properties of TGF-␤ receptors. In this report, it is shown that receptors undergo recycling both in the presence and absence of ligand activation, with the rates of internalization and recycling being unaffected by ligand binding. Recycling occurs as receptors are most likely internalized through clathrin-coated pits, and then returned to the plasma membrane via a rab11-dependent, rab4-independent mechanism. Together, the results suggest a mechanism wherein activated TGF-␤ receptors are directed to a distinct endocytic pathway for down-regulation and clathrin-dependent degradation after one or more rounds of recycling. INTRODUCTIONTGF-␤ is a ubiquitous 25-kDa polypeptide that regulates a variety of cellular processes, including matrix deposition, mitosis, development, differentiation, and apoptosis (Roberts, 1992;ten Dijke et al., 1996). The response to TGF-␤ treatment usually depends on the cell type involved, with effects as diverse as growth and growth inhibition (Massagué, 1996;Moses and Serra, 1996). TGF-␤ binds to singlepass transmembrane serine/threonine kinases referred to as type I and II TGF-␤ receptors (Bassing et al., 1994;ten Dijke et al., 1994). On binding of TGF-␤ to the constitutively active type II receptor (T2R), the type I receptor (T1R) is recruited and phosphorylated by T2R. The activated T1R then phosphorylates downstream signaling intermediates such as the Smad proteins, which translocate to the nucleus and function as transcriptional comodulators (Franzén et al., 1993;Macias-Silva et al., 1996;Yingling et al., 1996).Study of the endocytic response of TGF-␤ receptors to ligand has been difficult due to nonspecific TGF-␤ binding and the fact that different receptor complexes form on the cell surface (heteromers vs. homomers) and undergo distinct endocytic fates (Anders et al., 1997). To overcome these problems, our laboratory created a chimeric receptor system where the ligand binding extracellular domains of the granulocyte/macrophage-colony stimulating factor (GM-CSF) receptors were fused to the transmembrane and cytoplasmic domains of the type I and type II TGF-␤ receptors (Anders and Leof, 1996). Using a number of well-established assays for TGF-␤ action, it could be shown that TGF-␤ signaling is fully recapitulated by the chimeric system (Anders and Leof, 1996;Anders et al., 1997Anders et al., , 1998. In addition, use of the chimeric system (Anders et al., 1997) facilitated TGF-␤ endocytic assays, which showed, similar to other studies (Ehrlich et al., 2001;Yao et al., 2002), that ligand-induced internalization of TGF-␤ receptor complexes occurs through a clathrindependent process. Other reports, however, have proposed roles for...

Transforming growth factor-B (TGF-B) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-B in essentially all cell types, it is unlikely to be the sole mediator of cell type-specific outcomes to TGF-B stimulation. In the current study, we determined that TGF-B receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-B in vitro. (Cancer Res 2005; 65(22): 10431-40)

Members of the transforming growth factor ␤ (TGF-␤) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-␤ signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-␤ receptor (TGF-␤R) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-␤R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-␤-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-␤R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-␤R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.

Definitions based on a 50% or 75% decrease of CA 125 levels have been shown reliably to define partial response of ovarian cancer in patients receiving first-line chemotherapy. These definitions should be used in addition to or instead of standard response criteria.

SignificanceBoth highly pathogenic avian influenza virus and Middle East respiratory syndrome coronavirus (MERS-CoV) infections are characterized by severe disease and high mortality. The continued threat of their emergence from zoonotic populations underscores an important need to understand the dynamics of their infection. By comparing the host responses across other related respiratory virus infections, these studies have identified a common avenue used by MERS-CoV and A/influenza/Vietnam/1203/2004 (H5N1-VN1203) influenza to antagonize antigen presentation through epigenetic modulation. Overall, the use of cross-comparisons provides an additional approach to leverage systems biology data to identify key pathways and strategies used by viruses to subvert host immune responses and may be critical in developing both vaccines and therapeutic treatment.

Coronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2′O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2′O-MTase function across CoVs.

While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward.

Biological tissues exhibit complex spatial heterogeneity that directs the functions of multicellular organisms. Quantifying protein expression is essential for elucidating processes within complex biological assemblies. Imaging mass spectrometry (IMS) is a powerful emerging tool for mapping the spatial distribution of metabolites and lipids across tissue surfaces, but technical challenges have limited the application of IMS to the analysis of proteomes. Methods for probing the spatial distribution of the proteome have generally relied on the use of labels and/or antibodies, which limits multiplexing and requires a priori knowledge of protein targets. Past efforts to make spatially resolved proteome measurements across tissues have had limited spatial resolution and proteome coverage and have relied on manual workflows. Here, we demonstrate an automated approach to imaging that utilizes label-free nanoproteomics to analyze tissue voxels, generating quantitative cell-type-specific images for >2000 proteins with 100-µm spatial resolution across mouse uterine tissue sections preparing for blastocyst implantation.