Juan J. Carrillo

Coordinated agonist regulation of receptor and G protein palmitoylation and functional rescue of palmitoylation-deficient mutants of the G protein G11alpha following fusion to the alpha1b-adrenoreceptor: palmitoylation of G11alpha is not required for interaction with beta*gamma complex.

Transfection of either the α1b-adrenoreceptor or Gα11 into a fibroblast cell line derived from a Gαq/Gα11 double knockout mouse failed to produce elevation of intracellular [Ca2+] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the α1b-adrenoreceptor with the palmitoylation-resistant C9S,C10S Gα11 also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S Gα11 or C10S Gα11. Expression of a fusion protein between the α1b-adrenoreceptor and Gα11 allowed [Ca2+] i elevation, and this was also true for a fusion protein between the α1b-adrenoreceptor and C9S,C10S Gα11, since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin α, as a β·γ-sequestering agent, fully attenuated the Ca2+signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type Gα11 were also targets for agonist-regulated [3H]palmitoylation and bound [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [35S]GTPγS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release β·γ complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca2+ signaling in EF88 cells by such fusion proteins is mediated via release of the G protein β·γ complex.

G protein-coupled receptor fusion proteins in drug discovery

A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein alpha subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist/inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.

Monitoring receptor dimerisation

In recent times the concept that G protein-coupled receptors (GPCRs) can exist and potentially function as dimers or higher oligomers has become widely accepted. By considering GPCR-G protein fusion proteins as bi-functional polypeptides we have generated complementary pairs of non-functional mutants that are able to reconstitute function when co-expressed. Importantly for studies on the selectivity of GPCR hetero-dimerisation this allows development of assays in which the hetero-dimer, but not the co-expressed homo-dimers, are functional. The concept that GPCR hetero-dimers display distinct pharmacology can thus be tested directly.

CHAPTER 223 – Using Receptor-G-Protein Chimeras to Screen for Drugs

In the vast majority of cases analysis of GPCR-G protein chimeras takes place following expression in cell systems that also express endogenously the G protein of interest. A range of techniques has been developed to ensure that observed signals represent activation of the G protein within the chimera. Direct measures of the levels of expression of the α subunits of heterotrimeric G proteins indicate that they are generally in considerable excess over the levels of any particular G-protein-coupled receptor (GPCR) that might activate them. Despite this, an emerging strategy that has been used to address both basic questions on the details of GPCR-G protein interactions and to allow screens to be designed for agonist ligands at GPCRs has been to generate chimeric GPCR-G protein constructs that define a 1:1 stoichiometry of the two partner polypeptides. Such constructs provide a convenient means to assess the effects of mutations and polymorphisms in GPCRs on signal transduction effectiveness without altering the ratio of expression of GPCR to G protein. They have also allowed effective means of developing direct assays of GPCR-mediated guanine nucleotide exchange on G proteins of the G s and G q families that historically has been difficult to monitor. Such assays have allowed direct analysis of the extent of constitutive activity of different GPCRs and the detection of inverse agonists.