Receptor recycling mediates plasma membrane recovery of dopamine D1 receptors in dendrites and axons after agonist‐induced endocytosis in primary cultures of striatal neurons

Abstract

The pharmacological stimulation of G-protein-coupled receptor induces receptor internalization. Receptor's fate after the step of internalization remains poorly characterized despite its incidence on the neuronal responsiveness. In this context, we studied the dopamine (DA) D1 receptor (D1R) trafficking in a model of striatal neuronal culture that endogenously express the D1R. We first characterized by immunohistochemistry the spatial distribution of the compartments involved in the endocytic pathways and then the D1R trafficking in dendrites and axons. In dendrites, immunohistochemical analysis showed that acute stimulation by the D1R agonist SKF 82958 (1 microM) induces an internalization of D1R in early endosomes labeled with Alexa-488-conjugated transferrin. We show that, 20 min after removal of the agonist, the D1R immunolabeling pattern returns to the basal state in dendrites and in axons. Recovery was unaffected by cycloheximide (70 microM) but was prevented by monensin (100 microM) that inhibits endosomal acidification and receptor recycling. These data suggest that dendritic and axonal D1Rs are internalized after agonist stimulation and targeted to the recycling pathway demonstrating that the machinery involved in GPCR endocytosis and recycling is functional both in dendrites and in axons. Temporal characteristics observed for the recovery of D1R density to the basal state and those observed for the resensitization process strongly suggest that D1R recycling supports the receptor resensitization.

Human Immunodeficiency Virus (HIV) Infection of Human Macrophages Is Increased by Dopamine

“…Agonist concentrations were based on previous studies. 44,45 All MDMs (both infected and control cells) were treated with 200 mol/L ascorbic acid to prevent the formation of reactive oxygen species. 46 After 24 hours, MDMs were washed to remove viral inoculum, as well as DA, any agonists and ascorbic acid, and fed with fresh macrophage media.…”

Effects of veratridine and high potassium on μ-opioid receptor internalization in the rat spinal cord: Stimulation of opioid release versus inhibition of internalization

“…The mechanism of this inhibition probably involves blockade of MOR internalization by the high intracellular levels of Na + resulting from the opening of Na + channels by veratridine. The Na + ionophore monensin, which also promotes Na + entry into the cell, inhibits the recycling of internalized receptors (Martin-Negrier et al, 2006), but inhibition of receptor internalization by high intracellular Na + has not been reported. An alternative explanation is that 100 μM veratridine produced neurotoxic effects (Dargent et al, 1996;Pauwels et al, 1990).…”

l-DOPA Impairs Proteasome Activity in Parkinsonism through D1Dopamine Receptor

“…Four animals were randomly selected in each group for measuring proteasome catalytic activity. Primary medium spiny neuron culture: Sprague Dawley rat striatal cultures were prepared as previously described (Kowalski and Giraud, 1993;Martin-Negrier et al, 2000;Martin-Negrier et al, 2006). All the reagents for the culture were purchased from Sigma and Invitrogen.…”

“…In all other conditions, incubation media contained 0.1% of DMSO. For the agonist-induced internalization of D1R, cells were incubated with 10 M of the full D1R agonist SKF-82958 (Martin-Negrier et al, 2000;Martin-Negrier et al, 2006). To test the effect of the inhibition of proteasome activity on D1R trafficking, neurons were incubated with bortezomib for 60 min before 60 min incubation with SKF-82958.…”

“…a, In control condition, D1R immunofluorescent staining is punctated at the surface of the neurites. b, SKF-82958 treatment (60 min) reveals larger and brighter fluorescent spots in the neurites in a compartment corresponding to endosome (Martin-Negrier et al, 2006). c, Proteasome inhibition impairs this agonist-induced endocytosis, with reduced modifications of the density and the size of fluorescent puncta.…”