Ligand regulation of beta1- and beta2-adrenergic receptors and their GFP-tagged forms

G protein coupled receptors (GPCRs), as the name suggests, require a coupling transducer called a G protein to enable conversion of extracellular stimuli into intracellular second messengers. Over 200 GPCRs with the same topological seven transmembrane structure have been identified along with their coupling G proteins. To understand more about GPCR signalling and particular components required to achieve efficient signalling, extensive studies have involved the beta2-AR signalling pathway. Receptor activation and subsequent receptor desensitisation and internalisation of the beta2-AR and GPCRs in general have proved a major area of interest. Various types of ligand can bind to a GPCR such as agonist, inverse agonist and antagonist. Pharmacologists have struggled to clearly categorise these types of ligands. It was the aim of my first chapter to develop the basis for a GPCR ligand screen using the beta2-AR as a model system. Previously it has been demonstrated that a (constitutively active mutant) CAM-beta2-AR becomes up-regulated when treated with the inverse agonist, betaxolol for 24 h (MacEwan and Milligan, 1996a,b). In this study, a CAM-beta2-AR, C-terminally tagged with GFP was also found to be up-regulated by betaxolol. In fact a differential pattern of up-regulation was found to occur with a range of beta-blockers. The pattern of up-regulation was found to correlate with the ability of the ligands to induce production of cAMP, thus acting as partial agonists at the CAM-beta2-AR-GFP construct. This developed the basis for a rapid screening assay for ligand regulation of this receptor. Chapter 2 investigated the sequestration of the WT-beta1-AR, WT-beta2-AR and their GFP-tagged forms. It was shown that the beta1-AR constructs internalised more rapidly than the beta2-AR, but 10-20 % less maximal beta1-AR receptor was internalised compared to the beta2-AR. Tagging these receptors with GFP did not alter their pharmacology but receptor sequestration was markedly impeded, indicating limitations to the use of GFP as a tagging molecule. Down-regulation of the WT-beta2- AR-GFP was also smaller than that of the WT-beta2-AR after 24 h treatment of agonist stimulation. Receptor desensitisation and sequestration of the beta2-AR requires phosphorylation of the receptor by (protein kinase A) PKA and (GPCR kinases) GRKs. As the roles of these kinases and their sites of phosphorylation have been debated, a mutant form of the beta2-AR lacking all the potential C-terminal GRK phosphorylation sites (BARK-- beta2-AR-GFP) was used to investigate this issue. The construct was found to internalise in response to isoprenlaine stimulation, this being at a faster rate than internalisation of WT-beta2-AR-GFP. Secondly, the PKA inhibitor Rp cAMP had no effect on internalisation of this mutant construct, WT-beta2-AR or WT-P2-AR-GFP. These results suggest that PKA is not involved in sequestration of the beta2-AR and that other phosphorylation sites in the receptor may be responsible for this effect