Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors

GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.     

Interaction between GABAA receptor subunit intracellular loops: implications for higher order complex formation

The majority of fast inhibitory neurotransmission in the CNS is mediated by the GABA type-A (GABAA) receptor, a ligand-gated chloride channel. Of the approximately 20 different subunits composing the hetero-pentameric GABAA receptor, the gamma2 subunit in particular seems to be important in several aspects of GABAA receptor function, including clustering of the receptor at synapses. In this study, we report that the intracellular loop of the gamma2 subunit interacts with itself as well as with gamma1, gamma3 and beta1-3 subunits, but not with the alpha subunits. We further show that gamma2 subunits interact with photolabeled pentameric GABAA receptors composed of alpha1, beta2/3 and gamma2 subunits, and calculate the dissociation constant to be in the micromolar range. By using deletion constructs of the gamma2 subunit in a yeast two-hybrid assay, we identified a 23-amino acid motif that mediates self-association, residues 389-411. We confirmed this interaction motif by inhibiting the interaction in a glutathione-S-transferase pull-down assay by adding a corresponding gamma2-derived peptide. Using similar approaches, we identified the interaction motif in the gamma2 subunit mediating interaction with the beta2 subunit as a 47-amino acid motif that includes the gamma2 self-interacting motif. The identified gamma2 self-association motif is identical to the interaction motif reported between GABAA receptor and GABAA receptor-associated protein (GABARAP). We propose a model for GABAA receptor clustering based on GABARAP and GABAA receptor subunit-subunit interaction.     

Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor β (TGFβ). We have provided evidence that TGFβ regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRα1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFβ, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFβ. Additional treatment with TGFβ does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRα2 and GFRα1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRα1, TGFβ is not required for recruitment of GFRα2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFβ. Disruption of TGFβ signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRα1 crystallization and ontogenetic development of the CG that may explain the differences in TGFβ-dependence of GDNF and NRTN.     

Ligand Entry and Exit Pathways in the β2-Adrenergic Receptor

The recently determined crystal structure of the human β₂-adrenergic (β₂AR) G-protein-coupled receptor provides an excellent structural basis for exploring β₂AR-ligand binding and dissociation process. Based on this crystal structure, we simulated ligand exit from the β₂AR receptor by applying the random acceleration molecular dynamics (RAMD) simulation method. The simulation results showed that the extracellular opening on the receptor surface was the most frequently observed egress point (referred to as pathway A), and a few other pathways through interhelical clefts were also observed with significantly lower frequencies. In the egress trajectories along pathway A, the D192-K305 salt bridge between the extracellular loop 2 (ECL2) and the apex of the transmembrane helix 7 (TM7) was exclusively broken. The spatial occupancy maps of the ligand computed from the 100 RAMD simulation trajectories indicated that the receptor-ligand interactions that restrained the ligand in the binding pocket were the major resistance encountered by the ligand during exit and no second barrier was notable. We next performed RAMD simulations by using a putative ligand-free conformation of the receptor as input structure. This conformation was obtained in a standard molecular dynamics simulation in the absence of the ligand and it differed from the ligand-bound conformation in a hydrophobic patch bridging ECL2 and TM7 due to the rotation of F193 of ECL2. Results from the RAMD simulations with this putative ligand-free conformation suggest that the cleft formed by the hydrophobic bridge, TM2, TM3, and TM7 on the extracellular surface likely serves as a more specific ligand-entry site and the ECL2-TM7 hydrophobic junction can be partially interrupted upon the entry of ligand that pushes F193 to rotate, resulting in a conformation as observed in the ligand-bound crystal structure. These results may help in the design of β₂AR-targeting drugs with improved efficacy, as well as in understanding the receptor subtype selectivity of ligand binding in the β family of the adrenergic receptors that share almost identical ligand-binding pockets, but show notable amino acid sequence divergence in the putative ligand-entry site, including ECL2 and the extracellular end of TM7.     

A triple arg motif mediates α(2B)-adrenergic receptor interaction with Sec24C/D and export

Recent studies have demonstrated that cargo exit from the endoplasmic reticulum (ER) may be directed by ER export motifs recognized by components of the coat protein II (COPII) vesicles. However, little is known about ER export motifs and vesicle targeting of the G protein-coupled receptor (GPCR) superfamily. Here, we have demonstrated that a triple Arg (3R) motif in the third intracellular loop functions as a novel ER export signal for α(2B)-adrenergic receptor (α(2B)-AR). The 3R motif mediates α(2B)-AR interaction with Sec24C/D and modulates ER exit, cell surface transport and function of α(2B)-AR. Furthermore, export function of the 3R motif is independent of its position within α(2B)-AR and can be conferred to CD8 glycoprotein. These data provide the first evidence implicating that export of GPCRs is controlled by code-directed interactions with selective components of the COPII transport machinery.