Investigations into GABAb receptor surface stability and molecular interactions

Whereas most G-protein-coupled receptors (GPCRs) are monomeric in structure, y-Aminobutyric acid type B (GABAb) receptors are heterodimers of two seven transmembrane protein subunits, GABAB(i) and GABAB(2>. GABAb receptor function is dependent upon the co-expression of both these proteins which, when individually expressed, are devoid of receptor activity. Desensitisation of cell surface receptors allows tissues to rapidly adjust their response to agonist. A conserved mechanism ensures that GPCR signalling is closely followed by desensitisation. This entails the phosphorylation of activated receptors enabling interaction with arrestin proteins and subsequent internalisation. It is not known if heterodimeric GABAb receptors employ this method of desensitisation. Data presented here result from experiments to determine whether GABAb receptor cell surface stability is controlled in a similar manner to that of other GPCRs. Also documented is the study of a putative interaction between the protein kinase AMPK and the GABAB(i) subunit. Initial whole cell labelling studies demonstrated that both GABAB(i) and GABAB(2) are basally phosphorylated. Dissimilar to other GPCRs, agonist did not increase levels of phosphorylation and this remained true upon overexpression of G protein receptor kinases. Because GABAb receptors lacked the internalisation promoting increase in phosphorylation upon agonist, it was predicted that they might have enhanced surface stability. This was confirmed in heterologous systems where GABAb receptors did not demonstrably internalise after agonist application even when arrestins were overexpressed. GABAb receptors in cultured cortical neurones showed a similar lack of internalisation in response to agonist. Biotinylation of neuronal surface receptors demonstrated that GABAb receptors reside for an unusually long time at the plasma membrane. Chronic agonist decreased the surface receptor half-life, but this did not correlate with an increase in internalised receptor. Interestingly, chronic agonist did not significantly reduce total receptor protein levels, suggesting GABAb receptors may not downregulate. Protein kinase A