GABAB Receptor Association with the PDZ Scaffold Mupp1 Alters Receptor Stability and Function

Abstract

␥-Aminobutyric acid, type B (GABA B ) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABA B receptor (GABA B R) functionality, we screened the GAB-A B R2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABA B R2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABA B R2 in cells. Disruption of the GAB-A B R2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABA B receptor signaling. The effects of mutating the GABA B R2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABA B receptor stability and signaling can be modulated via GABA B R2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABA B receptors in the central nervous system. GABA B2 receptors are G protein-coupled receptors responsible for mediating slow inhibitory synaptic transmission by the neurotransmitter GABA (1). They belong to G protein-coupled receptor Family C and bear a high degree of homology to other family members such as the metabotropic glutamate receptors, calcium receptor, and vomeronasal receptors. GABA B receptors are believed to be heterodimeric combinations of two G protein-coupled receptors, GABA B R1 and GABA B R2 (2-4). Heterodimerization of GABA B R1 and GABA B R2 is necessary for the proper trafficking of GABA B R1 to the cell surface (5, 6). In the context of the heterodimer, GABA B R1 is thought to bind the ligand (7), whereas GABA B R2 is believed to be the primary G protein contact site (8 -11).Given that GABA B receptors are important therapeutic targets for a wide variety of diseases, including depression, anxiety, epilepsy, and drug addiction (12, 13), understanding GABA B receptor signaling and regulation is of significant clinical interest. The cloning of the GABA B receptors has advanced the study of the GABA B receptors substantially over the past decade. However, some discrepancies between the properties of native GABA B receptors and heterologously expressed recombinant receptors still remain. For example, GABA B receptors in native tissue undergo robust endocytosis and desensitization (14), whereas recombinant GABA B R1/GABA B R2 expressed in most heterologous cells neither internalize nor desensitize (14, 15). One possible explanation for such discrepancies is that GABA B receptor signaling and trafficking properties are highly dependent on cellular context. This implies that interaction with differentially expressed cellular proteins might modulate GABA B receptor function. Indeed, we...

Rapid modulation of spine morphology by the 5-HT 2A serotonin receptor through kalirin-7 signaling

“…In agreement with these previous studies, here we found that in cultured cortical neurons, 5-HT 2A receptors colocalize with both PSD-95 and MUPP1 in the dendrites and dendritic spines of cortical pyramidal neurons, and when coexpressed in COS-7 cells, MUPP1 facilitates the membrane targeting of 5-HT 2A receptors which can be prevented by mutating the receptor PDZ binding motif. Importantly, MUPP1 is a scaffold for a growing list of synaptic proteins that are involved in GPCR and small GTPase signaling (27,(39)(40)(41). Our data, along with previous studies, are consistent with the roles of both MUPP1 and PSD-95 as protein scaffolds that may integrate signals through GPCRs at the synapse, provide avenues for crosstalk between signaling pathways, and function in the membrane localization of some receptors and signaling proteins.…”

The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor

“…Effect of PDZ10 on MT 1 Internalization and DegradationInteraction of GPCRs with PDZ proteins has been shown to stabilize receptors by inhibiting either their constitutive or agonist-promoted internalization (8,34) or by interfering with receptor degradation (4,5). To study the role of MUPP1 in MT 1 internalization and degradation, we used the isolated PDZ10 domain of MUPP1 as a dominant negative to disrupt the MUPP1/MT 1 interaction.…”

“…G protein-coupled receptors (GPCRs) 5 constitute the largest family of membrane receptors, and many of the more than 750 members have been shown to interact with PDZ domain-containing proteins, either constitutively or upon agonist activation (2). Binding of PDZ proteins to GPCRs has been reported to primarily regulate subcellular localization, trafficking, and stability of receptors (3).…”

“…Binding of PDZ proteins to GPCRs has been reported to primarily regulate subcellular localization, trafficking, and stability of receptors (3). For instance, binding of MUPP1 and syntrophins to the ␥-aminobutyric acid type B (GABA B ) receptor and the ␣ 1D -adrenergic receptor, respectively, significantly increases receptor stability (4,5). In other cases, PDZ scaffolds determine the subcellular localization of GPCRs (6) and receptor endocytosis as shown for PSD-95 and the 5-HT 2A serotonin and ␤ 1 -adrenergic receptors (7,8).…”

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

“…Second, MAGI-2 overexpression was found to have no effect on ␤ 1 AR-mediated cAMP signaling and to promote agonist-induced ␤ 1 AR internalization (40), but it was found to reduce vasoactive intestinal polypeptide type-1 receptor-mediated cAMP signaling and suppress agonist-induced receptor internalization (41). Finally, although MUPP1 enhances GABA B receptor signaling, it functions to uncouple the melatonin-1 receptor from G␣ i (42,43). Similarly, this study demonstrates that SAP97 regulates CRFR1 trafficking, because overexpression of GFP-SAP97 prevents CRFR1 endocytosis and knockdown of endogenous SAP97 promotes CRFR1 endocytosis.…”