Heterotrimeric GTP-binding proteins (G proteins) control cellular functions by transducing signals from the outside to the inside of cells. Regulator of G protein signaling (RGS) proteins are key modulators of the amplitude and duration of G protein-mediated signaling through their ability to serve as guanosine triphosphatase-activating proteins (GAPs). We have identified RGS-PX1, a Galpha(s)-specific GAP. The RGS domain of RGS-PX1 specifically interacted with Galpha(s), accelerated its GTP hydrolysis, and attenuated Galpha(s)-mediated signaling. RGS-PX1 also contains a Phox (PX) domain that resembles those in sorting nexin (SNX) proteins. Expression of RGS-PX1 delayed lysosomal degradation of the EGF receptor. Because of its bifunctional role as both a GAP and a SNX, RGS-PX1 may link heterotrimeric G protein signaling and vesicular trafficking.
A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg2+ATP and Mg2+GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins α2p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of α2p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of α2p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPγS, inhibition by Brefeldin A (BFA), or depletion of β-COP from cytosol. Therefore, the p24 family member, α2p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the generation of ER cargo exit sites.
Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc Ϫ1 ) isolated from rat liver homogenates reconstitute tER by Mg 2ϩ GTP-and Mg 2ϩ ATP-hydrolysisdependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.
Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Gαs) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Gαs expression by RNA interference (RNAi) delays receptor degradation. We also show that Gαs and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Gαs coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Gαs colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Gαs on endosomes. Furthermore, knock-down of both Hrs and Gαs by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Gαs and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Gαs in EGF receptor degradation and provide mechanistic insights into the function of Gαs in endocytic sorting
Arginine-rich cell penetrating peptides are short cationic peptides able to cross biological membranes despite their peptidic character. In order to optimize their penetration properties and further elucidate their mechanisms of cellular entry, these peptides have been intensively studied for the last two decades. Although several parameters are simultaneously involved in the internalization mechanism, recent studies suggest that structural modifications influence cellular internalization. Particularly, backbone rigidification, including macrocyclization, was found to enhance proteolytic stability and cellular uptake. In the present work, we describe the synthesis of macrocyclic arginine-rich cell penetrating peptides and study their cellular uptake properties using a combination of flow cytometry and confocal microscopy. By varying ring size, site of cyclization, and stereochemistry of the arginine residues, we studied their structure-uptake relationship and showed that the mode and site of cyclization as well as the stereochemistry influence cellular uptake. This study led to the identification of a hepta-arginine macrocycle as efficient as its linear nona-arginine congener to enter cells.
The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and proteintyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV-(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.
LDL receptor-related protein 9 (LRP9) is a distant member of the low-density lipoprotein receptor (LDLR) superfamily. To date, there are no reports on the cellular distribution of LRP9 or the signals responsible for its localization. Here, we investigated the intracellular localization and trafficking of LRP9. Using confocal microscopy, we demonstrated that LRP9 was not present at the plasma membrane but co-localized with various markers of the trans-Golgi network (TGN) and endosomes. This co-localization was dependent on the presence of two acidic cluster/dileucine (DXXLL) motifs in the cytoplasmic tail of LRP9, which interact with GGA proteins, clathrin adaptors involved in transport between the TGN and endosomes. LRP9 is the first example of a transmembrane protein with an internal GGA-binding sequence in addition to the usual C-terminal motif. An inactivating mutation (LL --> AA) in both DXXLL motifs, which completely inhibited the interaction of LRP9 with GGA proteins, led to an intracellular redistribution of LRP9 from the TGN to early endosomes and the cell surface, indicating that the two DXXLL motifs are essential sorting determinants of LRP9. In conclusion, our results suggest that LRP9 cycles between the TGN, endosomes and the plasma membrane through a GGA dependent-trafficking mechanism.
Calnuc is an ubiquitous, EF-hand Ca(2+) binding protein found in the cytoplasm where it binds to Galphai3, in the Golgi lumen where it constitutes a Ca(2+) storage pool, and secreted outside the cell. Here we investigated the pathway of secretion of calnuc in AtT20 cells. We found by pulse-chase experiments that calnuc is synthesized in the endoplasmic reticulum, transported to the Golgi where it remains greater than 12 h and undergoes posttranslational modification (O-glycosylation and sulfation) followed by secretion into the culture medium. We examined if calnuc is secreted by the constitutive or regulated secretory pathway in AtT20 cells. By immunofluorescence and immunogold labeling, endogenous calnuc is found in immature secretion granules (ISG) but not mature regulated secretory granules (RSG), whereas overexpressed calnuc-green fluorescent protein (GFP) is found in both ISG and RSG, where it colocalizes with ACTH. Neither calnuc nor calnuc-GFP are released by the regulated secretory pathway, suggesting that endogenous calnuc and calnuc-GFP are progressively removed from ISG and RSG during granule maturation. We conclude that calnuc is secreted via the constitutive-like pathway and represents a useful endogenous marker for this pathway in AtT20 cells. Together, these observations indicate that calnuc has a unique itinerary as it is retained in the Golgi and is then constitutively secreted extracellularly where it may influence cell behavior via its Ca(2+)-binding properties.
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