Sushi domains confer distinct trafficking profiles on GABA _B receptors

Abstract

GABA B receptors mediate slow inhibitory neurotransmission in the brain and feature during excitatory synaptic plasticity, as well as various neurological conditions. These receptors are obligate heterodimers composed of GABA B R1 and R2 subunits. The two predominant R1 isoforms differ by the presence of two complement control protein modules or Sushi domains (SDs) in the N terminus of R1a. By using live imaging, with an α-bungarotoxin-binding site (BBS) and fluorophore-linked bungarotoxin, we studied how R2 stabilizes R1b subunits at the cell surface. Heterodimerization with R2 reduced the rate of internalization of R1b, compared with R1b homomers. However, R1aR2 heteromers exhibited increased cell surface stability compared with R1bR2 receptors in hippocampal neurons, suggesting that for receptors containing the R1a subunit, the SDs play an additional role in the surface stability of GABA B receptors. Both SDs were necessary to increase the stability of R1aR2 because single deletions caused the receptors to be internalized at the same rate and extent as R1bR2 receptors. Consistent with these findings, a chimera formed from the metabotropic glutamate receptor (mGluR)2 and the SDs from R1a increased the surface stability of mGluR2. These results suggest a role for SDs in stabilizing cell surface receptors that could impart different pre-and postsynaptic trafficking itineraries on GABA B receptors, thereby contributing to their physiological and pathological roles. underlying slow GABA-mediated inhibitory neurotransmission (1) that regulates neuronal excitability (2-4). These receptors are increasingly considered as potential therapeutic targets for a range of diseases, including epilepsy, schizophrenia, anxiety, depression, and substance abuse (1, 5). Functional GABA B receptors are heteromers formed from GABA B R1 subunits containing the agonist binding domain (6) and R2 subunits that link to G protein signaling (7,8).To date, just one isoform of the R2 subunit has been reported (9, 10), whereas several isoforms of the R1 subunit exist [R1a, R1b, R1c, R1e, R1j (1)]. Among these, R1a and R1b predominate in the central nervous system (CNS), and their expression arises by the use of different promoters (11) of the GABBR1 gene. R1a subunits contain 143 aa forming two Sushi domains (SDs) in the N terminus, which are also known as complement control proteins or short consensus repeats (12). These SDs are absent in R1b being replaced by 18 unique amino acids.Heteromers formed from R1aR2 and R1bR2 are thought to play distinct roles in neurotransmission (13-18), with R1aR2 contributing to presynaptic heteroreceptors to inhibit neurotransmitter release, although both R1aR2 and R1bR2 populate postsynaptic membranes to dampen excitability. These heteromeric subtypes exhibit different subcellular compartmentalization with the SDs acting as an axonal-targeting sequence to deliver R1aR2 more efficiently to axons compared with R1bR2 (19). This would, in part, explain the differential pre-and postsynaptic signaling properties o...

Cell surface expression of homomeric GABAA receptors depends on single residues in subunit transmembrane domains

“…The cells were imaged immediately post-fixation in saline using a Zeiss LSM 510 Meta confocal microscope and an Achroplan ×40 water differential interference contrast objective (numerical aperture (NA) 0.8) as described previously (48). This involved choosing the optimal z-section and acquiring individual images as a mean from 4 scans in 16 bits using a 543-nm helium–neon laser and a 560-nm long-pass filter for α-BgTx–AF555 and a 488 argon laser with a 505- to 530-nm band pass filter for eGFP.…”

GABA B(1) receptor subunit isoforms differentially regulate stress resilience

“…Unlike GABA B(1b) , GABA B(1a) can localize to axons via Sushi domains, which also increase surface stability of GABA B(1a,2) receptors (6,39). In dendrites, GABA B(1a) localizes to glutamatergic terminals for heteroceptor function whereas GABA B(1b) localizes to spines opposing glutamate release sites, thus affecting pre-or postsynaptic inhibition (6).…”

Identifying the role of pre-and postsynaptic GABAB receptors in behavior

“…Also known as a complement control protein, the presence of the sushi domain is believed to be responsible for the increased stability of B1a/B2 heterodimers as compared to B1b/B2 heterodimers. Further, inserting the B1a sushi domain into mGluR2, another Class III metabotropic receptor that has no endogenous sushi domain, increases surface stability (Hannan et al, 2012). As part of the complement control proteins, the sushi domains on B1a have long been proposed to be involved in protein-protein interactions that regulate the system (Marshall et al, 1999).…”

“…Both isoforms have been shown to act as autoreceptors, as baclofen reduces IPSC amplitude in both isoform knockouts to a similar extent (Vigot et al, 2006). On a larger systems level, Hannan et al (2012) suggest that the stability of the B1a receptor at presynaptic locations may act as an inhibitory brake, reducing excessive releases of glutamate. B1a receptors have been shown to gate glutamatergic inputs to excitatory neurons of the amygdala (Pan et al, 2009), and that presynaptic receptor activation from cortical afferents is important for LTP induction in lateral amygdala principal neurons (Lange et al, 2014).…”

“…Complement control protein 1, which is present only on B1a receptors, is able to bind to the matrix protein fibulin-2. Fibulin-2 can bind to extracellular ligands and calcium, but binding of fibulin-2 to the B1a receptor is not specific to the presence of complement control protein 1, indicating a non-specific effect (Hannan et al, 2012; Blein et al, 2004). Rajalu et al (2015) demonstrated that different K + channel tetramerization-domains, which bind to postsynaptic B1b/B2 receptors and the associated G-protein, do not alter basal GABA affinity or GABA affinity of native cells in the presence of the positive allosteric modulator GS39783.…”