GISP binding to TSG101 increases GABA_B receptor stability by down‐regulating ESCRT‐mediated lysosomal degradation

Abstract

1Both these authors contributed equally to this study.Abbreviations used: EGFR, epidermal growth factor receptor; ESCRT, endosomal sorting complex required for transport; GISP, G proteincoupled receptor interacting scaffold protein; GST, glutathione S-transferase; HEK293, human embryonic kidney cells. AbstractThe neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GA-BA B receptor subunit GABA B1 . Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA B2 subunit, their co-expression in HEK293 cells increases levels of GABA B2 . GISP also inhibits TSG101-dependent GABA B2 down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA B2 degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.

-Aminobutyric Acid Type B (GABAB) Receptor Internalization Is Regulated by the R2 Subunit

“…2C). Therefore, after being internalized, GABA B receptors are recruited to early endosomes, from where they are either recycled via recycling endosomes to be reinserted into the plasma membrane or are degraded in the lysosomes via the late endosomes, consistent with previous findings (14,15,27,28). The process of recycling would account for the plateau phase in the time profiles of surface fluorescence (Fig.…”

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) β-Dependent Phosphorylation of GABAB1 Triggers Lysosomal Degradation of GABAB Receptors via Mind Bomb-2 (MIB2)-Mediated Lys-63-Linked Ubiquitination

“…The two main cellular degradation systemsproteasomes and lysosomes-control the abundance of GABA B receptors in distinct cellular compartments in response to the activity state of the neuron [10,11,17]. In the ER, the amount of newly synthetized GABA B receptors destined for trafficking to the plasma membrane is regulated by proteasomal degradation via the ERAD machinery [10], whereas cell surface GABA B receptors are degraded in lysosomes [12][13][14][15][16]. Both degradation pathways require [10] and Lys-63-linked ubiquitination of GABA B1 at several sites sorts the receptors to lysosomes [17].…”

“…The activity state of the neuron controls the rate of Lys-48-linked ubiquitination of the GABA B2 subunit required for proteasomal receptor degradation [11]. In contrast, GABA B receptors internalized from the cell surface are degraded in lysosomes [12][13][14][15][16]. Sorting the receptors to lysosomes is mediated most likely via the endosomal sorting complex required for transport (ESCRT) machinery [16], which targets ubiquitinated membrane proteins to lysosomes.…”

“…In contrast, GABA B receptors internalized from the cell surface are degraded in lysosomes [12][13][14][15][16]. Sorting the receptors to lysosomes is mediated most likely via the endosomal sorting complex required for transport (ESCRT) machinery [16], which targets ubiquitinated membrane proteins to lysosomes. Accordingly, lysosomal degradation of GABA B receptors depends on Lys-63-linked ubiquitination of the GABA B1 subunit at multiple sites mediated by the E3 ubiquitin ligase MIB2 [17].…”

Endoplasmic Reticulum-associated Degradation Controls Cell Surface Expression of γ-Aminobutyric Acid, Type B Receptors

“…There is some evidence that GABA B receptors are sorted to lysosomes via the ubiquitin-dependent ESCRT (endosomal sorting complex required for transport) machinery (8). Hence, prolonged inhibition of proteasomes might indirectly compromise lysosomal degradation of the receptors.…”