PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles.

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.

The adaptor protein PID1 regulates receptor-dependent endocytosis of postprandial triglyceride-rich lipoproteins.

OBJECTIVE: Insulin resistance is associated with impaired receptor dependent hepatic uptake of triglyceride-rich lipoproteins (TRL), promoting hypertriglyceridemia and atherosclerosis. Next to low-density lipoprotein (LDL) receptor (LDLR) and syndecan-1, the LDLR-related protein 1 (LRP1) stimulated by insulin action contributes to the rapid clearance of TRL in the postprandial state. Here, we investigated the hypothesis that the adaptor protein phosphotyrosine interacting domain-containing protein 1 (PID1) regulates LRP1 function, thereby controlling hepatic endocytosis of postprandial lipoproteins. METHODS: Localization and interaction of PID1 and LRP1 in cultured hepatocytes was studied by confocal microscopy of fluorescent tagged proteins, by indirect immunohistochemistry of endogenous proteins, by GST-based pull down and by immunoprecipitation experiments. The in vivo relevance of PID1 was assessed using whole body as well as liver-specific Pid1-deficient mice on a wild type or Ldlr-deficient (Ldlr-/-) background. Intravital microscopy was used to study LRP1 translocation in the liver. Lipoprotein metabolism was investigated by lipoprotein profiling, gene and protein expression as well as organ-specific uptake of radiolabelled TRL. RESULTS: PID1 co-localized in perinuclear endosomes and was found associated with LRP1 under fasting conditions. We identified the distal NPxY motif of the intracellular C-terminal domain (ICD) of LRP1 as the site critical for the interaction with PID1. Insulin-mediated NPxY-phosphorylation caused the dissociation of PID1 from the ICD, causing LRP1 translocation to the plasma membrane. PID1 deletion resulted in higher LRP1 abundance at the cell surface, higher LDLR protein levels and, paradoxically, reduced total LRP1. The latter can be explained by higher receptor shedding, which we observed in cultured Pid1-deficient hepatocytes. Consistently, PID1 deficiency alone led to increased LDLR-dependent endocytosis of postprandial lipoproteins and lower plasma triglycerides. In contrast, hepatic PID1 deletion on an Ldlr-/- background reduced lipoprotein uptake into liver and caused plasma TRL accumulation. CONCLUSIONS: By acting as an insulin-dependent retention adaptor, PID1 serves as a regulator of LRP1 function controlling the disposal of postprandial lipoproteins. PID1 inhibition provides a novel approach to lower plasma levels of pro-atherogenic TRL remnants by stimulating endocytic function of both LRP1 and LDLR in the liver.

Sweet taste receptor interacting protein CIB1 is a general inhibitor of InsP3-dependent Ca2+ release in vivo.

In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Galpha(16gust44) and Galpha(15i3) link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca2+ concentration, we used sweet and umami compounds as well as other InsP3-generating ligands in FURA-2-based Ca2+ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca2+ response of HEK293 cells to the InsP3-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca2+ store of the endoplasmic reticulum (ER) and independent of extracellular Ca2+. The function of CIB1 on InsP3-evoked Ca2+ release from the ER is most likely mediated by its interaction with the InsP3 receptor. Thus, CIB1 seems to be an inhibitor of InsP3-dependent Ca2+ release in vivo.

The rat GPRC6A: cloning and characterization.

GPRC6A is a novel member of family C of G protein-coupled receptors with so far elusive biological function. GPRC6A has been described in human and mouse as a promiscuous l-alpha-amino acid receptor. We now report the cloning, expression analysis and, functional characterization of the rat orthologue of GPRC6A. Full-length cloning of rat GPRC6A (rGPRC6A) was accomplished using amplification of cDNA from taste tissue, and the identity of rGPRC6A confirmed at both the genomic and the protein level by similarity studies. Using selective primers, reverse transcriptase polymerase chain reaction showed that the mRNA is widely but weakly distributed, except for a high expression in the soft palate, the so-called geschmacksstreifen. On the protein level, rGPRC6A was shown to be glycosylated and most likely oligomeric, and using immunochemistry we observed that rGPRC6A is expressed at the plasma membrane of mammalian cell lines. Utilizing co-expression of rGPRC6A and the promiscuous Galpha(q)(G66D) protein in an engineered cell-based inositol phosphate turnover assay, we were able to study the ligand profile of the receptor. We found that l-ornithine is the most potent and efficacious l-amino acid agonist with an EC(50) value of 264 microM, followed by several other aliphatic, neutral, and basic amino acids. Furthermore, the divalent cation Mg(2+) was found to be a positive modulator of the l-ornithine response. The presented quantitative pharmacological data underlines the evolutionary conservation of GPRC6A to the rat and signifies the physiological importance and emerging pharmacological potential of GPRC6A.

Expression of different p63 variants in healing skin wounds suggests a role of p63 in reepithelialization and muscle repair.

Healing of skin wounds in mammals involves partial reconstruction of the dermis and coverage of the injured site by keratinocytes. The latter process is achieved by extensive migration and hyperproliferation of keratinocytes at the wound rim. Because the p53 protein family member p63 is expressed in human hyperproliferative epidermis, this study determined whether enhanced keratinocyte proliferation correlates with the expression of p63. Therefore, we investigated the temporal and spatial distribution of four major variants of the p63 transcription factor-TAp63alpha, TAp63gamma, DeltaNp63alpha and DeltaNp63gamma-during normal skin wound healing in mice. Transcripts encoding amino-terminally truncated DeltaNp63 variants were found at high levels in basal and suprabasal keratinocytes of the hyperproliferative wound epithelium. Interestingly, TAp63 variants, which include the conserved transactivation domain TA at their amino-terminus, were also expressed in wound keratinocytes as well as at the edge of the injured subcutaneous muscle panniculus carnosus. These findings suggest splice-variant specific functions of p63 in reepithelialization and muscle repair.

Rat inositol 1,4,5-trisphosphate 3-kinase C is enzymatically specialized for basal cellular inositol trisphosphate phosphorylation and shuttles actively between nucleus and cytoplasm.

The calcium-liberating second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) by Ins(1,4,5)P3 3-kinases (IP3Ks) that add a fourth phosphate group to the 3-position of the inositol ring. Two isoforms of IP3Ks (named A and B) from different vertebrate species have been well studied. Recently the cloning and examination of a human full-length cDNA encoding a novel isoform, termed human IP3K-C (HsIP3K-C), has been reported. In the present study we report the cloning of a full-length cDNA encoding a rat homologue of HsIP3K-C with a unique mRNA expression pattern, which differs remarkably from the tissue distribution of HsIP3K-C. Of the rat tissues examined, rat IP3K-C (RnIP3K-C) is mainly present in heart, brain, and testis and shows the strongest expression in an epidermal tissue, namely tongue epithelium. RnIP3K-C has a calculated molecular mass of approximately 74.5 kDa and shows an overall identity of approximately 75% with HsIP3K-C. A bacterially expressed, enzymatically active and Ca2+-calmodulin-regulated fragment of this isoform displays remarkable enzymatic properties like a very low Km for Ins(1,4,5)P3 ( approximately 0.2 microm), substrate inhibition by high concentrations of Ins(1,4,5)P3, allosteric product activation by Ins(1,3,4,5)P4 in absence of Ca2+-calmodulin (Ka(app) 0.52 microm), and the ability to efficiently phosphorylate a second InsP3 substrate, inositol 2,4,5-trisphosphate, to inositol 2,4,5,6-tetrakisphosphate in the presence of Ins(1,3,4,5)P4. Furthermore, the RnIP3K-C fused with a fluorescent protein tag is actively transported into and out of the nucleus when transiently expressed in mammalian cells. A leucine-rich nuclear export signal and an uncharacterized nuclear import activity are localized in the N-terminal domain of the protein and determine its nucleocytoplasmic shuttling. These findings point to a particular role of RnIP3K-C in nuclear inositol trisphosphate phosphorylation and cellular growth.

Differential regulation of transcription and induction of programmed cell death by human p53-family members p63 and p73.

The p53 tumor suppressor acts as a transcription factor and has a central function in controlling apoptosis. With p63 and p73 two genes coding for proteins homologous to p53 have been identified. We describe the properties of seven human p63 and p73 proteins as transcriptional activators of p21WAF1/CIP1 expression and apoptotic inducers in direct comparison to p53 in the same assay systems employing DLD-1-tet-off colon cells. Programmed cell death is detected in cells expressing high levels of p53 and p73alpha. Cells overexpressing TAp63alpha, TAp63gamma, TA*p63alpha, TA*p63gamma, DeltaNp63alpha, and DeltaNp63gamma display low or no detectable apoptosis.

Retinoic acid inhibits downregulation of DeltaNp63alpha expression during terminal differentiation of human primary keratinocytes.

Recently, the p53 homolog p63 has been implicated in sustaining the epidermal stem cell population. The p63 gene encodes six major products with transactivating or dominant-negative properties. The expression pattern of these isoforms in keratinocytes was investigated here. Northern blot, ribonuclease protection assay, reverse transcription-polymerase chain reaction, and western blot techniques sensitive for all six p63 isotypes verified the predominant expression of the truncated and potentially dominant-negative isotype DeltaNp63alpha in human keratinocytes. The expression of this isoform is downregulated when proliferating human primary keratinocytes begin to differentiate after growth factor withdrawal. The onset of differentiation does not change the ratio of two other weakly expressed isotypes DeltaNp63gamma and TAp63alpha relative to DeltaNp63alpha. Treatment of primary human keratinocytes with all-trans retinoic acid does not alter the expression pattern of p63 isotypes but prevents its downregulation as observed in control cell cultures. These data suggest that p63 expression in human keratinocytes is affected by all-trans retinoic acid and this influence might contribute to the fine tuned keratinocyte proliferation and differentiation equilibrium in the mammalian epidermis.