Reciprocal recruitment of DRIP/mediator and p160 coactivator complexes in vivo by estrogen receptor

Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter.     

The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.     

The nuclear receptor coactivator PGC-1alpha exhibits modes of interaction with the estrogen receptor distinct from those of SRC-1

Estrogen receptor (ER) function is mediated by multi-domain co-regulator proteins. A fluorescently labelled fragment of the human PGC-1alpha co-regulator (residues 91-408) bearing the two motifs most strongly implicated in interactions with nuclear receptors (NR box2 and NR box3), was used to characterize in vitro binding of PGC-1alpha to ER. Anisotropy measurements revealed that the affinity of this PGC-1alpha fragment for human ERalpha and beta was fairly strong in the presence of estradiol (approximately 5 nM), and that unlike a similar fragment of SRC-1 (570-780), PGC-191-408 exhibited ligand-independent interactions with ER, particularly with ERbeta (Kd approximately 30 nM). Competition experiments of the complex between ERalpha and fluorescently labelled PGC-1 91-408 with unlabelled SRC-1 570-780 showed that PGC-1 91-408 was an efficient competitor of SRC-1 570-780, while the inverse was not true, underscoring their distinct modes of binding. The anisotropy data provide strong evidence for a ternary complex between ERalpha, SRC-1 570-780 and PGC-1 91-408. GST-pull-down experiments with deletion mutants of ERalpha revealed that the constitutive binding of PGC-1 91-408 requires the presence of the linker domain between the DNA binding and ligand binding domains (DBD and LBD). Homology modeling studies of the different regions of full length PGC-1alpha confirmed the lack of compact tertiary structure of the N-terminal region bearing the NR box motifs, and suggested a slightly different mode of interaction compared to the NR box motifs of SRC-1. They also provided reasonable structural models for the coiled-coil dimerization motif at residues 633-675, as well as the C-terminal putative RNA binding domain, raising important questions concerning the stoichiometry of its complex with the nuclear receptors.     

The function of TIF2/GRIP1 in mouse reproduction is distinct from those of SRC-1 and p/CIP

Human TIF2 (hTIF2) is a member of the p160 family of nuclear receptor coactivators, which includes SRC-1 and p/CIP. Although the functions of hTIF2 and of its mouse homolog (GRIP1 or mTIF2) have been clearly established in vitro, their physiological role remains elusive. Here, we have generated mice lacking mTIF2/GRIP1 and examined their phenotype with a particular emphasis on reproductive functions. TIF2(-/-) mice are viable, but the fertility of both sexes is impaired. Male hypofertility is due to defects in both spermiogenesis (teratozoospermia) and age-dependent testicular degeneration, and TIF2 expression appears to be essential for adhesion of Sertoli cells to germ cells. Female hypofertility is due to a placental hypoplasia that most probably reflects a requirement for maternal TIF2 in decidua stromal cells that face the developing placenta. We conclude that TIF2 plays a critical role in mouse reproductive functions, whereas previous reports have not revealed serious fertility impairment in SRC-1(-/-) or p/CIP(-/-) mutants. Thus, even though the three p160 coactivators exhibit strong sequence homology and similar activity in assays in vitro, they play distinct physiological roles in vivo, as their genetic eliminations result in distinct pathologies.     

The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains

Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD mu1A or mu1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B-containing membranes that were both distinct from AP-1A-positive TGN elements and more closely apposed to transferrin receptor-positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.