Hannan, S.B.; (2011) Cell surface mobility of GABAB receptors. Doctoral thesis , UCL (University College London).

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Abstract

Type-B γ-aminobutyric acid receptors (GABABRs) are important for mediating slow inhibition in the central nervous system and the kinetics of their internalisation and lateral mobility will be a major determinant of their signalling efficacy. Functional GABABRs require R1 and R2 subunit co-assembly, but how heterodimerisation affects the trafficking kinetics of GABABRs is unknown. Here, an α-bungarotoxin binding site (BBS) was inserted into the N-terminus of R2 to monitor receptor mobility in live cells. GABABRs are internalised via clathrin- and dynamin-dependent pathways and recruited to endosomes. By mutating the BBS, a new technique was developed to differentially track R1a and R2 simultaneously, revealing the subunits internalise as heteromers and that R2 dominantly-affects constitutive internalisation of GABABRs. Notably, the internalisation profile of R1aR2 heteromers, but not R1a homomers devoid of their ER retention motif (R1ASA), is similar to R2 homomers in heterologous systems. The internalisation of R1aASA was slowed to that of R2 by mutating a di-leucine motif in the R1 C-terminus, indicating a new role for heterodimerisation, whereby R2 subunits slow the internalization of surface GABABRs. R1a and R1b are the predominant GABABR1 isoforms in the brain, differing by the two Sushi Domains (SDs) in R1a. Introduction of a BBS into the N-terminus of R1b and comparison with R1a revealed that R1bR2 internalises faster than R1aR2. Introduction of the SDs into the BBS-tagged metabotropic glutamate receptor-2 also conferred a decrease in internalisation. Finally, the lateral surface mobility of GABABRs was studied by extending the BBS-tagging method to single-particle tracking using quantum dots. R1aR2 and R1bR2 exhibited different mobility profiles on hippocampal neurons and differentially responded to baclofen. In conclusion, this study provides new and important insight into the mobility of cell surface GABABRs and the underlying mechanisms that ensure they provide efficacious slow synaptic inhibition.

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