Heterodimeric coiled-coil interactions of human GABA _B receptor

Abstract

Metabotropic GABA B receptor is a G protein-coupled receptor that mediates inhibitory neurotransmission in the CNS. It functions as an obligatory heterodimer of GABA B receptor 1 (GBR1) and GABA B receptor 2 (GBR2) subunits. The association between GBR1 and GBR2 masks an endoplasmic reticulum (ER) retention signal in the cytoplasmic region of GBR1 and facilitates cell surface expression of both subunits. Here, we present, to our knowledge, the first crystal structure of an intracellular coiled-coil heterodimer of human GABA B receptor. We found that polar interactions buried within the hydrophobic core determine the specificity of heterodimer pairing. Disruption of the hydrophobic coiled-coil interface with single mutations in either subunit impairs surface expression of GBR1, confirming that the coiled-coil interaction is required to inactivate the adjacent ER retention signal of GBR1. The coiled-coil assembly buries an internalization motif of GBR1 at the heterodimer interface. The ER retention signal of GBR1 is not part of the core coiled-coil structure, suggesting that it is sterically shielded by GBR2 upon heterodimer formation.

Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis

“…The GABA B1 subunit contains an ER retention motif in the C‐terminal tail that inhibits its trafficking to the cell surface when expressed alone. Co‐expression of GABA B1 with GABA B2 masks the ER retention motif of GABA B1 , allowing both to traffic to the cell surface where they exist as a stable di‐sulphide linked heteromer . As expected, HiBiT‐GABA B1 was poorly expressed alone (though intracellular expression could be detected with the addition of digitonin) and co‐expression of GABA B2 greatly enhanced the expression of HiBiT‐GABA B1 at the cell surface as measured by HiBit luminescence (Figure B).…”

Allosteric interactions between GABAB1 subunits control orthosteric binding sites occupancy within GABAB oligomers

“…GABA B1 binds GABA in its VFT and GABA B2 plays a major role in G protein activation (Galvez et al, 2001;Jones et al, 1998;Kaupmann et al, 1998;White et al, 1998). The heterodimers are stabilized by a coiled-coil interaction involving the intracellular C-termini as well as additional noncovalent interactions between their VFTs and 7TMs (Burmakina et al, 2014;Geng et al, 2013;Kammerer et al, 1999;Kuner et al, 1999). The coiled-coil interaction directly participates in a quality control of heterodimerization since GABA B1 is retained in the endoplasmic reticulum (ER) unless the ER retention signal present in its C-terminus is masked through the coiled-coil interaction with GABA B2 allowing the heterodimer to reach the plasma membrane (Calver et al, 2001;Margeta-Mitrovic et al, 2000;Pagano et al, 2001).…”

Signaling via GABAB receptors regulates early development and neurogenesis in the basal metazoan Nematostella vectensis

“…These included a signal peptide, followed by a large extracellular Nterminal domain that contains the structural VFT module that binds GABA, as well as seven trans-membrane (TM) domains that are characteristic of G protein-coupled receptors. Predicted coiled-coil motifs, which are found in the human GABABR where they mediate heterodimerization and receptor trafficking (41,42), were predicted in only three Nematostella homologs. In general, the intracellular C-terminus portion of the Nematostella homologs had low similarity to the corresponding human sequences.…”