ABSTRACT
N regions at the junction of V, D and J DNA segments are synthesized with large protein complexes including terminal deoxynucleotidyltransferase (TdT) during V(D)J recombination in B- or T-cells. TdT directly binds to TdIF1, TdIF2, PCNA and the Ku70/86 heterodimer. Using a yeast two-hybrid system, we isolated a cDNA clone encoding the gene for TReP-132, which is involved in P450scc gene expression in steroid-hormone-producing cells or lymphoid cells. Interaction between TReP-132 and TdIF1 was confirmed by pull-down assay and immunoprecipitation assay using specific antibodies against TReP-132 both in vitro and in vivo. TdT also directly bound to TReP-132 through its confined N-terminal region. Furthermore, the co-expression of TdIF1 and TReP-132 or TdT and TReP-132 in COS7 cells showed that these proteins are co-localized within the nucleus. TReP-132 reduces TdT activity to 2.5% of its maximum value in the in vitro assay system using double-stranded DNA with a 3' protrusion as a primer. These findings suggest that TdT synthesizes N region under a negative control of TReP-132 during V(D)J recombination.
Subject(s)
DNA Nucleotidylexotransferase/metabolism , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Animals , COS Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Chlorocebus aethiops , DNA/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/analysis , Down-Regulation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Swine , Thymus Gland/chemistry , Thymus Gland/cytology , Transcription Factors/analysis , Two-Hybrid System TechniquesABSTRACT
The cell-surface localization of GPCRs (G-protein-coupled receptors) has emerged as one of critical factors of the GPCR-mediated signal transduction. It has been reported that the C-termini of GPCRs contain the sequences for sorting the receptors to cell surface. In the present study, we have searched for proteins that interact with the C-terminus of PTH (parathyroid hormone)/PTH-related protein receptor (PTHR) by using the yeast two-hybrid system, and identified a cytoskeletal protein 4.1G (generaltype 4.1 protein) as an interactant with the C-terminus. Immunohistochemical study revealed that both PTHR and 4.1G were co-localized on plasma membranes, when they were transiently expressed in COS-7 cells. When 4.1G or the C-terminal domain of 4.1G (4.1G-CTD), a dominant-negative form of 4.1G, was co-expressed with PTHR in COS-7 cells, 4.1G, but not 4.1G-CTD, facilitated the cell-surface localization of PTHR, determined by cell-surface biotinylation assay. PTH-(1-34) caused phosphorylation of ERK (extracellular-signal-regulated kinase) 1/2 in PTHR-expressed cells mainly mediated through EGF (epidermal growth factor) receptor. The phosphorylation was enhanced by the expression of 4.1G, but not 4.1G-CTD. PTH-(1-34) elevated [Ca2+]i (intracellular Ca2+ concentration) independent of EGF receptor activation, and the elevation was enhanced by the expression of 4.1G, but not 4.1G-CTD. These data indicate that 4.1G facilitates the cell-surface localization of PTHR through its interaction with the C-terminus of the receptor, resulting in the potentiation of PTHR-mediated signal transduction.
Subject(s)
Cytoskeletal Proteins/metabolism , Membrane Proteins/metabolism , Receptor, Parathyroid Hormone, Type 1/metabolism , Animals , Calcium/metabolism , Cell Line , Cell Membrane/metabolism , ErbB Receptors/metabolism , Gene Expression Regulation , Humans , MAP Kinase Kinase Kinases/metabolism , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Protein Transport , Two-Hybrid System TechniquesABSTRACT
COOH-terminal cytoplasmic domains of G protein-coupled receptors (GPCRs) have been shown to carry determinants that control their cell surface localization, internalization, and recycling. In attempts to seek cellular proteins that mediate these processes of PTH/PTH-related protein receptor (PTHR), one of the class B GPCRs, we have found that Tctex-1, a 14kDa light chain of cytoplasmic dynein motor complex, interacts with the COOH-terminal tail of the receptor. A 34-amino-acid stretch of the receptor responsible for binding to Tctex-1 has a bipartite structure consisting of a motif previously implicated in binding of some proteins to Tctex-1 and a putative new consensus sequence. Site-directed mutations or a 20-amino-acid deletion in the bipartite consensus binding sequence abolished the association of the PTHR COOH terminus with Tctex-1 in vitro. A GFP-fused mutant PTHR impaired in binding to Tctex-1 expressed in MDCK cells showed a decreased rate of internalization in response to PTH compared to that of the wild type.
