G protein-coupled receptors (GPCRs) are cell surface proteins that translate hormone or ligand binding into intracellular signals. GPCRs are found in all animals, insects, and plants. GPCR signaling plays a pivotal role in regulating various physiological functions including phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, digestion, pain, and fluid and electrolyte balance. Although they are involved in various physiological functions, GPCRs share a number of common structural features. They contain seven membrane domains bridged by alternating intracellular and extracellular loops and an intracellular carboxyl-terminal tail of variable length.
The magnitude of the physiological responses controlled by GPCRs is linked to the balance between GPCR signaling and signal termination. The signaling of GPCRs is controlled by a family of intracellular proteins called arrestins. Arrestins bind GPCRs, including those that have been agonist activated and bind more tightly to those that have been phosphorylated by G protein-coupled receptor kinases (GRKs) than those that are not.
Receptors, including GPCRs, have historically been targets for drug discovery and therapeutic agents because they bind ligands, hormones, and drugs with high specificity. Approximately fifty percent of the therapeutic drugs in use today target or interact directly with GPCRs. See e.g., Jurgen Drews, (2000) “Drug Discovery: A Historical Perspective,” Science 287:1960-1964.
Although only several hundred human GPCRs are known, it is estimated that more than a thousand GPCRs exist in the human genome. Of these known GPCRs, many are orphan receptors that have yet to be associated with a function or specific ligands.
There is a continuing need for increasingly more accurate, easy to interpret methods of detecting G protein-coupled receptor activity and methods of assaying GPCR activity. One method, as disclosed in Barak et al., U.S. Pat. Nos. 5,891,646 and 6,110,693, uses a cell expressing a GPCR and a conjugate of an arrestin and a label molecule, the contents of which patents are incorporated by reference in their entirety.
In some instances, naturally occurring GPCRs do not provide optimal conditions for association with arrestin for easy detection. Accordingly, for those receptors that do not exhibit optimal conditions for association with arrestin, there is a need to increase affinity of the naturally occurring GPCRs with arrestin to provide for a more sensitive assay. Two distinct patterns of arrestin trafficking within the cell have been delineated resulting in the classification of GPCRs as follows: Class A (e.g. β2AR, α1b adrenergic receptor, μ opioid receptor, endothelin1A and dopamine D1A receptors) where arrestin interacts with the receptor at the cell surface but does not endocytose into vesicles, thus showing a transient interaction with the receptor, and class B (e.g. V2R, angiotensin AT1a, neurotensin1, thyrotropin releasing hormone and neurokinin NK-1 receptors) in which β-arrestins and receptor traffic together from the cell membrane to endocytic vesicles. These two classes of receptors also differ with regard to their affinity for different arrestin isoforms. In addition, Class A receptors preferentially bind β-arrestin2 whereas class B receptors bind to β-arrestin1 and β-arrestin2 with equal affinity.
β-arrestin-binding leads to the uncoupling of the receptor from its cognate G-proteins, causing dampening or desensitization of GPCR signaling via the downstream second messenger molecules. Recently, novel adaptor and scaffold functions of arrestins have been discovered. Thus, while terminating G-protein signals, arrestin binding can initiate new signalwaves from GPCRs. For example, β-arrestins serve as adaptors, which bring nonreceptor tyrosine kinases such as Src, to form signaling complexes with the internalizing receptor. β-arrestins function as GPCR-regulated scaffolds for MAPK modules such as ASK-MKK4-JNK3 and RAF-MEKERKI/2. In addition, arrestins interact with proteins of the endocytic machinery, such as clathrin, β-adaptin subunit2 of the AP2 complex, and Arf-6 and thus promote internalization of receptors via clathrin-coated vesicles.
Ubiquitination, in vivo, is a post-translational attachment of one or more ubiquitin molecules to the lysines of substrate proteins has been implicated to play a role in the internalization of yeast pheromone receptors and in the endocytosis of several mammalian cell-surface receptors. The prototypic mammalian GPCR, β2AR is also ubiquitinated in an agonist and β-arrestin-dependent manner. It appears that receptor ubiquitination is not crucial for its internalization but is essential for proper trafficking to lysosomes for degradation. On the other hand, β2AR internalization requires the agonist promoted ubiquitin modification of the adaptor protein β-arrestin2 catalyzed by a RING domain containing E3-ubiquitin ligase Mdm2.
Ubiquitin is a 76-amino acid residue monomeric protein so named because it is abundant in all eukaryotes and very highly conserved from yeast to humans. There are very few amino acid residue differences from species to species in the various ubiquitins in nature. Ubiquitin is normally associated with the degradation pathway of cytosolic proteins in proteosomes. The covalent binding of ubiquitin to a protein is normally the first step in marking a protein for degradation. Ubiquitination of a protein occurs by an energy dependent process that transfers ubiquitin from an ubiquitin-protein ligase (E3) to the E-amino group of a lysine on the target protein. Normally, the isopeptide bond formed between the lysine of a protein to be degraded and ubiquitin can be removed by appropriate peptidases. Polyubiquitin chains can often form on a protein via isopeptide bonds at a Lys (usually LYS 48) of ubiquitin and the C-terminal carboxyl group of the following ubiquitin. Additionally, attachment of ubiquitin at LYS 63 of ubiquitin is implicated to play a role in ubiquitin dependant endocytosis. It is clear therefore; that it would be useful to improve the binding of arrestin to a GPCR in order to improve the arrestin mediated detection of activated or inhibited GPCRs and in one embodiment that GPCR would be a Class A GPCR. In U.S. Ser. No. 09/993,844 incorporated herein by reference, a method of improving the binding affinity of arrestin and a GPCR is disclosed involving modifying the GPCR by genetically changing the sequence of the carboxy terminal tail of the GPCR to include a higher number of phosphorylation sites. This is an extremely useful technique but does require the modification of each GPCR requiring improved binding affinity and such modification are not ideal because they have the potential to change the ligand binding properties of the receptor.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of detecting G protein-coupled receptor activity, it should be apparent that there still exists a need in the art for methods to improving the binding affinity of arrestin to GPCRs without the need to make modifications to each individual GPCR.