The N-terminal domain of GABA receptor subunit ρ1 contains regions required for subunit assembly

Purpose. The retina GABA_C-like receptor subunit ρ1 forms robustly expressing homooligomeric receptors, whereas the GABA_A receptor subunits require heterooligomerization for efficient expression. This indicates that each ρ1 subunit has the information for precise assembly into a functional receptor. This study was performed to investigate the topology of ρ1 and to localize the domain directing subunit assembly. Methods. Two ρ1 mutants were created: N-ρ1, containing a termination signal at codon 256 immediately prior to the putative first transmembrane domain, and C-ρ1, in which sequence from the putative signal peptide to the first hydrophobic region was deleted. Wild-type ρ1 and the truncation mutants were translated in vitro for immunoprecipitation and protease susceptibility studies, using rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomes (Promega). For functional analysis, cRNA was synthesized from each mutant, injected into oocytes and current amplitudes were recorded. Results. N-ρ1 was resistant to digestion by proteinase K, demonstrating that it was translocated into the lumen of the microsomal vesicles. In contrast, ρ1 and C-ρ1 were reduced in size, indicating that a portion of these proteins remain at the exterior of the vesicle. Antibody M2, which recognizes a synthetic epitope on ρ1 but not N-ρ1, immunoprecipitated N-ρ1 when it was cotranslated with ρ1, indicating that they interact. Coinjection of Xenopus oocytes with wild-type ρ1 and N-ρ1 cRNA reduced ρ1 current in a dose-dependent manner: a 2:1 ratio of N-ρ1 to ρ1 decreased current to 10% of maximal (n=5), and a 5:1 ratio eliminated currents entirely (n=5). Coinjection of C-ρ1 and ρ1 cRNA did not inhibit ρ1 currents at any ratio. Conclusion. These results predict that the N-terminus of ρ1 is extracellular and a significant portion of the C-terminus is intracellular. Immunoprecipitation confirms that N-p1 interacts with ρ1, and expression studies demonstrate that N-ρ1 interfers with the formation of functional ρ1 receptors. Together, these data indicate that the N-terminus of ρ1 contains sequences important for assembly of homooligomeric ρ1 receptors.