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    • Two critical determinants of receptor trafficking

    2024-04-29

    Two critical determinants of receptor trafficking are found within the GABAB1 cytoplasmic tail: the di-leucine internalization signal (EKSRLL) (Margeta-Mitrovic et al., 2000, Restituito et al., 2005) and the ER retention signal (RSRR) (Calver et al., 2001, Margeta-Mitrovic et al., 2000, Pagano et al., 2001). The former reduces the level of GABAB1 expression on the cell surface, while the latter completely eliminates it (Margeta-Mitrovic et al., 2000). The internalization sequence lies within a coiled-coil region in the intracellular domain (Fig. 1A). It is directly blocked by the formation of a coiled-coil heterodimer between the GABAB1 and GABAB2 subunits as shown by the heterodimer crystal structure (Burmakina et al., 2014) (Fig. 3). The retention signal does not exist inside the coiled-coil region itself, but rather a few residues away from the C-terminal end of the motif. Nevertheless, mutations of the coiled-coil interfacial residues in either subunit prevent GABAB1 from expressing on the surface (Burmakina et al., 2014, Margeta-Mitrovic et al., 2000). Although the heterodimeric coiled-coil interface does not seal the retention signal, it prevents access to the motif through steric hindrance (Burmakina et al., 2014).
    Signaling complex GABAB receptor is unique in possessing auxiliary KCTD subunits to generate functional Roflumilast australia in neurons (Gassmann and Bettler, 2012). Four different KCTD molecules (numbered 8, 12, 12b and 16) affect GABAB receptor activity (Bartoi et al., 2010, Schwenk et al., 2010). All of these KCTD proteins are constitutively bound to the intracellular domain of GABAB2 subunit, and expedite agonist-dependent receptor activation by pre-anchoring the G protein to the GABAB receptor TM domain (Schwenk et al., 2010, Turecek et al., 2014). Aside from this function, each KCTD has its own role within the GABAB receptor signaling complex (Gassmann and Bettler, 2012). Within seconds after receptor activation, KCTD12 and KCTD12b attach to the βγ subunits of G protein, and disengage the structures from their target GIRK channel (Turecek et al., 2014). This action desensitizes the receptor and ceases hyperpolarization of the neuron (Schwenk et al., 2010, Seddik et al., 2012, Turecek et al., 2014). In addition, KCTD8 decreases basal G protein activation and agonist-independent GABAB receptor signaling (Rajalu et al., 2015). Finally, KCTD16 provides a template for binding effector channels such as the hyperpolarization-activated cyclic nucleotide gated (HCN) channels (Schwenk et al., 2016). Three-dimensional structures of GABAB receptor-associated KCTD proteins are not yet known. Once available, the structural information will provide important insights on how KCTD subunits allosterically modulate agonist affinity, G protein activation, and receptor desensitization.
    Activation mechanism Like their class A counterparts (Manglik and Kobilka, 2014), class C GPCRs exist in a conformational equilibrium between resting and active states in the absence of ligands (Kniazeff et al., 2011, Pin and Bettler, 2016). Agonists drive the equilibrium toward active states, while antagonists enhance the inactive states. The ensemble of conformations can be further stabilized by various allosteric modulators. Activation is associated with conformational transitions within the receptor structure (Fig. 3). In this section, we discuss the similarities and differences in the conformational states of various class C GPCRs. For all class C GPCRs, the first step of receptor activation involves agonist-triggered closure of the extracellular VFT module. For GABAB receptor, this event only occurs in the ligand-binding subunit, GABAB1 (Geng et al., 2013). In the case of mGlu receptors, closure of one protomer is sufficient to induce the active conformation, although full activation requires both VFT modules of the homodimer to be occupied and closed (Kniazeff et al., 2004, Kunishima et al., 2000, Tsuchiya et al., 2002). The significance of dual-VFT closure on mGlu receptor activation is further demonstrated in mGlu2-mGlu4 heterodimers, where mutations that prevent agonist binding to either subunit only result in partial activation (Moreno Delgado et al., 2017). Structural evidence also suggests that the active state of CaS receptor homodimer (Geng et al., 2016, Zhang et al., 2016) and TAS1R2-TAS1R3 heterodimer (Nuemket et al., 2017) involve both their protomers in the closed conformation.