Fig. 2.

GASP modulates D2R trafficking. (A) GASP (Top) coimmunoprecipitates selectively with FLAG-tagged D2Rs (Middle, compare D1R and D2R lanes). (Bottom) Lysate samples immunoblotted for GASP. HEK, HEK293 cells with no additional receptor; IP, immunoprecipitate; IB, immunoblot. (B) Cells were generated that stably overexpress GFP-cGASP. Lysates from cells stably expressing D2R only (left lane, undiluted lysate) or stably expressing both D2R and GFP-cGASP (all other lanes, lysate serially diluted 2-, 4-, 8-, or 80-fold) were immunoblotted for GASP. In the D2R-cGASP cell line, cGASP is >80-fold more abundant than endogenous GASP. (C) Cells stably expressing D2R were transiently transfected with GFP-cGASP. Cells were then treated with dopamine for 30 min (DA30) to monitor endocytosis, or dopamine for 30 min followed by agonist washout and antagonist haloperidol treatment for 30 min (DA30 + H30) to monitor recycling. D2Rs in adjacent cells that did (gray stars) or did not (white arrows) receive GFP-cGASP endocytosed in response to dopamine (Left). D2Rs in cells that overexpressed cGASP were recycled in 30 min (gray stars, Right). (D) Cells stably expressing FLAG-D2R (Upper) and stably overexpressing GFP-cGASP (Lower) were treated with dopamine for 90 min (DA90) to monitor endocytosis (Left) or with dopamine for 90 min, followed by a haloperidol (H) chase to monitor recycling (Right). D2Rs in these cells were recycled (compare to cells with D2R alone Fig. 1 A). (E) Cells from B either stably overexpressing HA-tagged D2R alone (Upper) or overexpressing both GFP-cGASP and HA-tagged D2R (Lower) were monitored for postendocytic trafficking by using the BPA. D2R degradation is attenuated by overexpression of GFP-cGASP (compare Upper and Lower DA120 and Q120 lanes). The D2Rs that were not degraded in the presence of GFP-cGASP were recycled within 30 min (compare DA120 to DA90/H30 and Q120 to Q90/H30 in the GFP-cGASP/D2R cell line, Lower).