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Investigations into GABAB receptor surface stability and molecular interactions.

Fairfax, B.P.; (2004) Investigations into GABAB receptor surface stability and molecular interactions. Doctoral thesis , University of London. Green open access

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Whereas most G-protein-coupled receptors (GPCRs) are monomeric in structure, gamma-Aminobutyric acid type B (GABAB) receptors are heterodimers of two seven transmembrane protein subunits, GABAB(1) 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(1) subunit. Initial whole cell labelling studies demonstrated that both GABAB(1) 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 (PKA) stimulation, both exogenously and through intracellular pathways, counteracted the agonist-induced degradation and demonstrated that this particular kinase can control GABAB receptor surface numbers. Protection from degradation was correlated with increased phosphorylation at serine 892 within GABAB(2), a residue previously demonstrated to be a PKA substrate. Subsequent experiments were carried out to identify kinases capable of phosphorylating GABAB(1). Affinity purification assays isolated a kinase from brain able to interact with and phosphorylate a twenty amino acid stretch of the carboxy-terminal domain of GABAB(1). Yeast two-hybrid studies identified the catalytic subunit of AMPK as a putative interacting protein with GABAB(1). AMPK was found to phosphorylate GABAB(1) at serines 917 and 923 within the carboxy-terminal domain. Phosphorylation of serine 917 was further confirmed with a phospho-specific antibody raised to this residue. AMPK affinity purifies with GABAB(1) carboxy-terminal domain GST fusion proteins and also co-immunoprecipitates with GABAB receptors from brain. Preliminary investigations indicate that AMPK activation increases surface GABAB receptor levels in cortical neurones and may affect the protein protein interactions of GABAB(1). The results presented in this thesis suggest GABAB receptors are highly stable at the neuronal surface. The activation of PKA and AMPK may be mechanisms by which neurones are able to regulate plasma membrane GABAB receptors.

Type: Thesis (Doctoral)
Title: Investigations into GABAB receptor surface stability and molecular interactions.
Identifier: PQ ETD:602750
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL
URI: https://discovery.ucl.ac.uk/id/eprint/1446808
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