GABAB receptor modulation by interacting protein

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABAA receptors, which are chloride-permeant ion channels, and GABAB receptors, which signal through coupling to G proteins. GABAB receptors are believed to be heterodimeric combinations of two G protein-coupled receptors, GABABR1 and GABABR2. In this dissertation work, I describe physical interactions that my colleagues and I have found between GABA B receptors and two different classes of proteins. The first of these is the association between GABAB receptor, GABABR1 with the GABAA receptor gamma2S subunit. This association promotes cell surface expression of GABABR1 in the absence of GABA BR2, the closely-related GABAB receptor subtype that is usually required for efficient trafficking of GABABR1 to the cell surface. The GABABR1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates with the functional GABABR1/GABABR2 heterodimer to significantly enhance GABAB receptor internalization in response to agonist stimulation.;The second novel association described here is between GABAB receptors and PDZ scaffold proteins. In order to identify novel interacting partners that might regulate GABAB receptor functionality, we screened the GABABR2 carboxyl-terminus against a proteomic array of 96 distinct PDZ (PSD-95/Drosophila Discs Large/ZO1 homology) domains. This screen identified three specific PDZ domains that exhibit interactions with GABABR2: Mupp1 PDZ13, PAPIN PDZ1 and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABA BR2 in cells. Disruption of the GABABR2 interaction with PDZ scaffolds by a point mutation to the receptor's carboxyl-terminus dramatically decreased receptor stability and also attenuated the duration of GABAB receptor signaling. The effects of mutating the GABA BR2 carboxyl-terminus on receptor stability and signaling were mimicked by siRNA knockdown of endogenous Mupp1. These findings reveal that GABA B receptor trafficking, signaling and stability can be differentially modulated by GABAB receptor interactions with the GABAA receptor gamma2S subunit and the PDZ scaffold protein Mupp1. These interactions may contribute to cell type-dependent regulation of GABAB receptors in the central nervous system and the fine tuning of inhibitory neurotransmission mediated by GABA.