Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface

Abstract

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.

Figure 1. Interaction between the D2R and TRPC proteins

(A) Results from a yeast two-hybrid screen showing interaction between TRPC1 and the D2IC2 domain (1) but not with an unrelated bait (D2IC3L; 3) or protein 4.1N (4). Interaction between CAPS and the D2IC2 domain (2) was included as a positive control. (B) The D2R associates with TRPC1. D2IC2-GST fusion protein pulled down S-tagged TRPC1 (residues 638–759) from a bacterial lysate. Only very faint bands were detected when the lysate was absorbed onto beads alone (control lane) or beads containing GST protein (GST lane). (C) The D2R also interacts with TRPC4 and TRPC5 proteins. The D2IC2 fusion protein pulled down S-tagged TRPC4 (residues 621–893; upper panel) and TRPC5 (residues 619–973; lower panel) from bacterial lysates. Only very faint immunoreactive bands were detected when lysates were absorbed onto beads containing GST protein (GST lane).

Figure 2. Localization of the D2R binding site on TRPC1, TRPC4 and TRPC5

Schematic representation of constructs encoding truncations of TRPC1. (A) Full-length TRPC1 protein. Shaded boxes depict ankyrin repeats (ANK), shaded ovals represent transmembrane (TM) segments, and black box depicts the pore loop domain (PL). Residues 694–759 encompass the C-terminal fragment (Clone 44) identified in the original yeast two-hybrid screen. (B) TRPC1 truncation fragments A–E were tested for interaction with the D2IC2 domain in the two-hybrid assay. Interaction is indicated by the presence (+) or absence (−) of β-galactosidase activity. (C) Amino acid sequence alignment of TRPC1, TRPC4, and TRPC5 in the C-terminal region found to contain the D2IC2 binding site. Amino acids are numbered to the left and right of each line. Identical amino acids are highlighted in black, and conserved amino acids are highlighted in grey. (D) Representative β-galactosidase assays comparing the interaction of D2IC2 with Clone 44 (residues 694–759 of TRPC1; 1), TRPC1 (residues 726–745; 2), TRPC4 (residues 712–731; 3), and TRPC5 (residues 719–738; 4). Interaction between TRPC1 (residues 726–745) and the D2LIC3 is shown as a negative control (5).

Figure 3. TRPC1 Interacts with D2R in Rat Cortex and Striatal Tissue

Anti-D2R antibodies were used to immunopreciptate D2Rs from rat cortex and striatum (ac-Abcam; sc-Santa Cruz; cb-Calbiochem). Blots containing immunocomplexes (IP) were sequentially probed with anti-TRPC1 antibodies (antibody used in top and middle panels was the monoclonal anti-TRPC1 antibody from Leonidas Tsiokas, University of Oklahoma; antibodies used in bottom panel were from Sigma). The position of TRPC1 endogenously expressed in cortical and striatal tissue is shown (lysate lane). No signal was observed in lysate sample adsorbed onto beads alone (Mock IP)

Figure 4. TRPCs interact with D2R and other components of the D2R signalplex

(A) Anti-D2R antibody (Abcam) was used to immunopreciptate D2Rs from cortex and striatum of rat. Blots containing immunocomplexes were sequentially probed with anti-TRPC1 (monoclonal anti-TRPC1 antibody) and anti-TRPC4 (Chemicon) antibodies. The positions of TRPC1 and TRPC4 endogenously expressed in cortical and striatal tissue is shown (lysate lanes). No signal was seen in lysate adsorbed onto beads alone (Mock IP). (B) NCS-1-GST fusion protein was used to pull down S-tagged TRPC1 (residues 638–759) from a bacterial lysate. S-tagged TRPC1 was pulled down in the presence of NCS-1-GST, but not with GST or beads alone. The position of the TRPC truncation fragment is shown in the lysate lane.

Figure 5. Expression profile of TRPC1 and D2Rs in neurons of the PFC

(A) Immunolabeling of TRPC1 subunits in the perikarya and dendrites of cortical neurons. TRPC1 immunolabeling was found in association with the (granular and agranular) endoplasmic reticulum and along the trans-Golgi axis (large inset). A limited plasmalemmal expression was also found at or near sites of juxtaposition of smooth endomembranes (small inset). (B) D2R expression was predominantly localized to (extrasynaptic ) membrane-bound compartments of high-order dendritic branches (den), and virtually absent in nearby axo-spinous (ax-sp) synapses. Scale bars: (A) 1 μm; (A-insets) 200 nm; (B) 400 nm.

Figure 6. Colocalization of D2R and TRPC1 in the cortex

(A–C) TRPC1 labeled with immunoperoxidase is shown in dendritic branches expressing both cytoplasmic and plasmalemmal D2R-immunoparticles (arrowheads). Note that the diffuse TRPC1 labeling in A (indicated by bracket within frame) is restricted to a portion of the dendrite and overlaps with the expression of D2Rs at the plasma membrane (frame). Other profiles reactive for D2R alone are seen in A and C. To test for reagent and method selectivity, we reversed the immunocytochemical sequence. In D, D2R labeling with immunoperoxidase overlaps with TRPC1 immunoparticles (double arrowheads) within membranes of intracellular compartments of a primary dendrite (lower inset). In E and C, dual nanogold labeling for D2Rs (large particles; arrowheads) and TRPC1 (small particles; double arrowheads) shows that immunoprobes are often separated by fewer than 50 nm. (F) Spatial colocalization of D2R and TRPC1 immunoparticles strongly suggests that the two proteins are physically associated at the plasma membrane of dendrites. Asterisks mark a subsurface cistern; ax, axon; den, dendrite; sp, spine. Scale bars: 200 nm.

Figure 7. TRPC1/D2R interaction mediates cell surface expression of TRPC1

A cell surface biotinylation assay was used to examine the effect of the D2R on plasma membrane expression of TRPC1. HEK293, HEK293-D2L, and HEK293-MOR cell surface proteins were biotinylated by incubation with NHS-SS- biotin. Biotinylated membrane proteins were immunoprecipitated using an anti-TRPC1 antibody (Alomone) and TRPC1 proteins quantitated by densotimetric analysis. (A) Anti-TRPC1 antibody was used to immunoprecipitate TRPC1 proteins from HEK293 and HEK293-D2L cells. Immunoblots were probed for the presence of biotin to identify cell surfaced- expressed TRPC1. (B) Total expression of TRPC1 in crude membrane fractions prepared from HEK293 and HEK293-D2L cells. (C) Biotinylated TRPC1 proteins in A were quantitated by laser densitometry. (D) Anti-TRPC1 antibody was used to immunoprecipitate TRPC1 from HEK293-MOR and HEK293-D2L cells. (E) Total expression of TRPC1 in HEK293-D2L and HEK293-MOR cells. (F) Biotinylated TRPC1 proteins in D were quantitated by laser densitometry. HEK293-D2L cells stably expressing D2Rs show an approximately 50% increase in the level of cell surface TRPC1 proteins (Students two-tailed t test, n=6, *p< 0.05) compared to either HEK293 cells or HEK293-MOR cells (n=4. *p<0.05). (G) Anti-TRPC1 antibody was used to immunoprecipitate TRPC1 proteins from HEK293-D2L and HEK293-MOR cells. Blots containing TRPC1 immunocomplexes were probed with anti-FLAG antibodies to identify D2L-FLAG and MOR-FLAG proteins. D2R and TRPC1 coimmunoprecipitated from HEK293-D2L cells, whereas TRPC failed to coimmunoprecipitate with the MOR expressed in HEK293-MOR cells.