γ-Aminobutyric Acid Type B Receptors Are Constitutively Internalized via the Clathrin-dependent Pathway and Targeted to Lysosomes for Degradation

Abstract

Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of ␥-aminobutyric acid, type B (GABA B ) receptors. Therefore, we analyzed internalization of GABA B receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40 -50% of cell surface receptors was detected. Stimulation of GABA B receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonistinduced internalization. The data suggest that GABA B receptors were endocytosed via the classical dynamin-and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABA B receptors via clathrincoated pits, which resulted in lysosomal degradation of the receptors. GABA B3 receptors are G protein-coupled receptors that play an important role in the control of neurotransmission. They are widely expressed in the nervous system and have been implicated as potential targets for neurological diseases, such as epilepsy, pain, spasticity, addiction, schizophrenia, depression, and anxiety (for a review, see Ref. 1). GABA B receptors mediate slow inhibitory neurotransmission by either activating postsynaptically K ϩ channels or inhibiting presynaptically the release of neurotransmitters by modulation of Ca 2ϩ channels. On the structural level, functional GABA B receptors require the heterodimerization of two distinct seven-transmembrane proteins, termed GABA B1 and GABA B2 (2-7). Two main variants of GABA B1 have been reported (GABA B1a and GABA B1b (8)), which are generated by alternative promoter usage (9) and differ solely in their N-terminal domain. Heterodimerization of GABA B1a or GABA B1b with GABA B2 leads to two main GABA B receptor subtypes, GABA B1a /GABA B2 and GABA B1b /GABA B2 , which are abundantly expressed in all major brain structures (10 -13).An important aspect in the regulation of G protein-coupled receptors is their internalization or endocytosis. To protect cells against receptor overstimulation, the vast majority of G protein-coupled receptors desensitize upon prolonged agonist exposure, followed by rapid internalization. Many G protein-coupled receptors undergo phosphorylation upon agonist exposure by a G protein receptor kinase and subsequently recruit an arrestin protein (14). Arrestins often enhance phosphorylation, sterically interfere with binding of the G protein, and function as a signal for receptor endocytosis (15)...

Constitutive, agonist-accelerated, recycling and lysosomal degradation of GABAB receptors in cortical neurons

“…This finding is in line with so far published data obtained under similar experimental conditions irrespective whether receptors were expressed in neurons (Fairfax et al, 2004;Vargas et al, 2008) or in heterologous expression systems (Fairfax et al, 2004;Grampp et al, 2007;Mutneja et al, 2005;Perroy et al, 2003). These experiments suggest that chronic activation does not promote endocytosis of GABA B receptors.…”

“…This extent of GABA B receptor internalization in neurons was considerably less than that we previously observed using GABA B receptors transiently expressed in HEK293 cells (Grampp et al, 2007). In HEK 293 cells, internalized receptors were already detectable after 10 min and after 120 min about 40-50% of cell surface receptors were found to be endocytosed.…”

“…One potential limitation of this assay for the analysis of GABA B receptor endocytosis in neurons was that the extent of internalized receptors expressed in HEK 293 cells critically depended on the antibody concentration used for labeling cell surface receptors. High antibody concentrations considerably inhibited endocytosis of GABA B receptors (Grampp et al, 2007).…”

“…Previously, we had shown that GABA B receptors heterologously expressed in HEK 293 cells display constitutive clathrin-dependent internalization and subsequent degradation in lysosomes using primarily antibody-based internalization assays (Grampp et al, 2007). One potential limitation of this assay for the analysis of GABA B receptor endocytosis in neurons was that the extent of internalized receptors expressed in HEK 293 cells critically depended on the antibody concentration used for labeling cell surface receptors.…”

“…At present, the precise mechanisms and pathways of GABA B receptor internalization remain to be determined. We have recently shown that GABA B receptors heterologously expressed in HEK 293 cells are constitutively internalized via the clathrin-dependent pathway and are targeted to lysosomes for degradation (Grampp et al, 2007). Neither agonist-induced internalization nor recycling of GABA B receptors was observed.…”