Functional Regulation of Dopamine D₃ Receptor through Interaction with PICK1

PICK1, a PDZ domain-containing protein, is known to increase the reuptake activities of dopamine transporters by increasing their expressions on the cell surface. Here, we report a direct and functional interaction between PICK1 and dopamine D₃ receptors (D₃R), which act as autoreceptors to negatively regulate dopaminergic neurons. PICK1 colocalized with both dopamine D₂ receptor (D₂R) and D₃R in clusters but exerted different functional influences on them. The cell surface expression, agonist affinity, endocytosis, and signaling of D₂R were unaffected by the coexpression of PICK1. On the other hand, the surface expression and tolerance of D₃R were inhibited by the coexpression of PICK1. These findings show that PICK1 exerts multiple effects on D₃R functions.

In vitro expression and analysis of the 826 human G protein-coupled receptors

G protein-coupled receptors (GPCRs) are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.

Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes.

CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

INFLUENCE OF PARTIALLY C-TERMINAL DELETION OF NR2A SUBUNITS ON THE SURFACE EXPRESSION AND FUNCTION OF NMDA RECEPTOR CHANNELS / 解剖学报

Objective To investigate whether the partially C-terminal deletion of NR2A subunit alters the surface expression and channel function of NMDA receptors in both HEK293 cells and cultured hippocampal neurons of rats. Methods Four plasmids for NR2A mutants with N-terminally GFP-tagged and C-terminal deletion NR2A?C1(?897L-1017S),NR2A?C2(?1024D-1142P),NR2A?C3(?1149D-1347G),and NR2A?C4(?1354S-1464V) were generated,and transfected into HEK293 cells and hippocampal neurons in culture.Surface staining was performed using anti-GFP antibody and Cy3 conjugated secondary antibody.Glutamate evoked currents were also detected using whole-cell recording. Results Positive surface staining was found for all the HEK293 cell co-transfected NR1-1a/NR2A?C1,NR1-1a/NR2A?C2,NR1-1a/NR2A?C3 or NR1-1a/NR2A?C4,and quantitative analysis showed no significant decrease in surface expression level when compared to that from NR1-1a/NR2A transfection.Glutamate-evoked currents were recorded in HEK293 cells co-transfected with NR1-1a/NR2A?C2 or NR1-1a/NR2A?C4.Surface expression level of NMDA receptor clusters on dendrites was significantly decreased in the neurons transfected with NR2A?C1,NR2A?C2 or NR2A?C3 than in those transfected with NR2A.Conclusion C-terminal deletion of NR2A subunit differentially effects surface expression of NMDA receptors in HEK293 cells and in hippocampal neurons in culture.