Cushing's disease is caused by corticotroph adenomas of the pituitary. To explore the molecular mechanisms of endocrine autonomy in these tumors, we performed exome sequencing of 10 corticotroph adenomas. We found somatic mutations in the USP8 deubiquitinase gene in 4 of 10 adenomas. The mutations clustered in the 14-3-3 protein binding motif and enhanced the proteolytic cleavage and catalytic activity of USP8. Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling. USP8 mutants enhanced promoter activity of the gene encoding proopiomelanocortin. In summary, our data show that dominant mutations in USP8 cause Cushing's disease via activation of EGF receptor signaling.

We present the design parameters, production process, and in-flight performance of the X-ray telescope (XRT) onboard Suzaku. The imaging capability is significantly improved over the ASCA XRT, which had half-power diameters of ${3\rlap {.}{}^{\mathrm {\prime }}6}$, to ${1\rlap {.}{}^{\mathrm {\prime }}8}$–${2\rlap {.}{}^{\mathrm {\prime }}3}$ for all four XRT-I modules. The optical axes are found to be distributed within a radius of ${1\rlap {.}{}^{\mathrm {\prime }}3}$, which makes the observation efficiency of all the XRTs more than 97% at the XIS-default observing position. The vignetting over the XIS field of view predicted via ray-tracing coincides with that measured for observations of the Crab Nebula to within $\sim 10\%$. Contemporaneous fits of a power law to all of the XIS spectra of the Crab Nebula taken at the two standard observing positions (XIS/HXD-default positions) gives a flux consistent with that obtained by Toor and Seward (1974, AJ, 79, 995) to within $\sim 2\%$. The pre-collimator on the top of each XRT module successfully reduces the intensity of the stray light from the $20'$ and $50'$-off directions down to the level of pre-flight expectations.

The biological function of receptors is determined by their appropriate trafficking through the endosomal pathway. Following internalization, the transferrin (Tf) receptor quantitatively recycles to the plasma membrane, whereas the epidermal growth factor (EGF) receptor undergoes degradation. To determine how Tf and EGF engage these two different pathways we imaged their binding and early endocytic pathway in live cells using total internal reflection fluorescence microscopy (TIRF-M). We find that EGF and Tf bind to distinct plasma membrane regions and are incorporated into different endocytic vesicles. After internalization, both EGF-enriched and Tf-enriched vesicles interact with endosomes containing early endosome antigen 1 (EEA1). EGF is incorporated and retained in these endosomes, while Tf-containing vesicles rapidly dissociate and move to a juxtanuclear compartment. Endocytic vesicles carrying EGF recruit more Rab5 GTPase than those carrying Tf, which, by strengthening their association with EEA1-enriched endosomes, may provide a mechanism for the observed cargo-specific sorting. These results reveal pre-endocytic sorting of Tf and EGF, a specialized role for EEA1-enriched endosomes in EGF trafficking, and a potential mechanism for cargo-specified sorting of endocytic vesicles by these endosomes.

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The FYVE domain is a conserved protein motif characterized by its ability to bind with high affinity and specificity to phosphatidylinositol 3-phosphate (PI(3)P), a phosphoinositide highly enriched in early endosomes. The PI(3)P polar head group contacts specific amino acid residues that are conserved among FYVE domains. Despite full conservation of these residues, the ability of different FYVE domains to bind to endosomes in cells is highly variable. Here we show that the endosomal localization in intact cells absolutely requires structural features intrinsic to the FYVE domain in addition to the PI(3)P binding pocket. These features are involved in FYVE domain dimerization and in interaction with the membrane bilayer. These interactions, which are determined by non-conserved residues, are likely to be essential for the temporal and spatial control of protein associations at the membrane-cytosol interface within the endocytic pathway.The FYVE domain is a double zinc finger named after the first four proteins found to contain this motif (Fab1, YotB, Vac1p, and EEA1) (1). FYVE domains bind with high specificity to phosphatidylinositol 3-phosphate (PI(3)P) 1 (2-5), which is highly enriched in early endosomes (3). Crystallographic and NMR analyses of isolated FYVE domains and crystallographic analysis of the homodimeric C terminus of EEA1 (6 -10) have revealed that six residues from the FYVE domain directly contact the Ins(1,3)P 2 group. These residues are localized within three conserved signature motifs, the WXXD, R(R/K)H-HCR, and RVC motifs.Although FYVE domains bind PI(3)P efficiently in vitro, when expressed in cells, isolated FYVE domains often fail to localize to endosomes (11)(12)(13)(14). Observations such as these have suggested that the ability of FYVE domains to localize to early endosomes may be determined by structural features distinct from those directly involved in PI(3)P binding (10). However, there is very little sequence conservation among FYVE domains in regions that do not directly contact the Ins(1,3)P 2 group. Thus, the molecular basis for the differing ability of FYVE domains to localize to endosomes remains unknown.To resolve this question, we have systematically analyzed the cellular localization of a series of isolated FYVE domains from the mammalian proteins EEA1, Hrs, SARA, FGD1, and frabin. Beyond the FYVE domain, there are no functional or structural similarities among these proteins, and they each have been hypothesized to fulfill distinct cellular functions (13,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).With the exception of one residue in the FYVE domain of FGD1, all these FYVE domains contain the specific residues that directly contact PI(3)P, and all bind to PI(3)P in vitro. However, as shown here, the ability of these domains to interact with endosomes when expressed in intact cells varies greatly. A comparative analysis of the structural features within these FYVE domains that correlate with in vivo endosome binding reveals a crucial role for two variable...