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Constitutively translocating cell line

Constitutively translocating cell line description/claims

The present application is a Continuation Application of U.S. patent application Ser. No. 10/788,197, filed on Feb. 26, 2004, which is a Continuation-In-Part application of International Application No. PCT/US03/14581, filed on May 12, 2003, which claims the benefit of U.S. Provisional Application No. 60/379,986 filed on May 13, 2002; and U.S. Provisional Application No. 60/401,698 filed on Aug. 7, 2002; which are hereby incorporated by reference in their entireties.

The present invention relates to methods of assaying GPCR desensitization in a agonist-independent manner, host cells useful in such methods, methods of the identification of compounds that alter GPCR desensitization, the compounds identified, and their use in disease treatment.

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 numerous 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.

GPCRs have been implicated in a number of disease states, including, but not limited to: cardiac indications such as angina pectoris, essential hypertension, myocardial infarction, supraventricular and ventricular arrhythmias, congestive heart failure, atherosclerosis, renal failure, diabetes, respiratory indications such as asthma, chronic bronchitis, bronchospasm, emphysema, airway obstruction, upper respiratory indications such as rhinitis, seasonal allergies, inflammatory disease, inflammation in response to injury, rheumatoid arthritis, chronic inflammatory bowel disease, glaucoma, hypergastrinemia, gastrointestinal indications such as acid/peptic disorder, erosive esophagitis, gastrointestinal hypersecretion, mastocytosis, gastrointestinal reflux, peptic ulcer, Zollinger-Ellison syndrome, pain, obesity, bulimia nervosa, depression, obsessive-compulsive disorder, organ malformations (for example, cardiac malformations), neurodegenerative diseases such as Parkinson's Disease and Alzheimer's Disease, multiple sclerosis, Epstein-Barr infection and cancer.

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 activated GPCRs, including those that have been agonist-activated and especially those that have been phosphorylated by G protein-coupled receptor kinases (GRKs).

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.

There is a need for 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 detectable molecule, the contents of which are incorporated by reference in their entirety.

Although only several hundred human GPCRs are known, it is estimated that upwards of a thousand GPCRs exist in the human genome. Of these known GPCRs, many are orphan receptors that have yet to be associated with a ligand.

The majority of the existing methods for identifying GPCR antagonists are dependent on the presence of agonist. Assays for identifying compounds that prevent the activation of GPCRs typically require that the GPCR is first activated in order to identify interfering compounds. For receptors with known agonists, these agonists are currently used to activate these receptors. However, many GPCRs are orphan receptors with no known ligand or agonist.

The agonist-dependence of GPCR assays continues to be a problem because antagonist discovery for orphan receptors is typically dependent on the prior discovery of agonist or ligand. Agonist-independent methods to screen for compounds that alter GPCR desensitization will (1) eliminate the step of agonist-addition in screening methods, and (2) enable identification of compounds that alter the desensitization of orphan receptors. Agonist-independent methods will eliminate the step of identifying an agonist of an orphan receptor prior to screening for compounds that alter desensitization of the orphan receptor.

The present invention relates to methods of identifying compounds which alter GPCR internalization.

A first aspect of the present invention is a method of identifying a compound which alters GPCR internalization, including: (a) providing a cell including a GPCR, an arrestin, and a modified GRK, wherein said GPCR is at least partially internalized in an agonist-independent manner upon expression of said GRK; (b) exposing said cell to the compound(s); (c) determining the cellular distribution of the GPCR, arrestin, or modified GRK; and (d) monitoring a difference between (1) the distribution of the GPCR, arrestin, or modified GRK in the cell in the presence of the compound(s) and (2) the distribution of the GPCR, arrestin, or modified GRK in the cell in the absence of the compound(s). An agonist may not be provided in the above method. In the method, a difference between (1) and (2) of step (d) may indicate modulation of GPCR internalization.

The GRK may be over-expressed, its expression may be inducible, and it may include a CAAX motif. The GRK may be GRK1, GRK2, GRK3, GRK4, GRK5, GRK6, or a biologically active fragment thereof.

The GPCR may be modified to have enhanced phosphorylation by a GRK. The GPCR may be β2AR(Y326A), a GPCR listed in FIG. 1A-1C, an orphan GPCR, a modified GPCR, a taste receptor, a Class A GPCR, a Class B GPCR, a mutant GPCR, or a biologically active fragment thereof.

The arrestin may be visual arrestin, cone arrestin, β-arrestin 1, β-arrestin-2, or a biologically active fragment thereof.

The GPCR, GRK, or arrestin may be detectably labeled. A molecule involved in desensitization may be detectably labeled, or a molecule that interacts with a molecule involved in desensitization may be detectably labeled.

In a further aspect, the present invention relates to a method of identifying a compound that alters GPCR phosphorylation, including: (a) providing a cell including a GPCR and a GRK; (b) exposing the cell to the compound(s); and (c) determining whether GRK phosphorylation of the GPCR is altered in the presence of the compound(s).

The cellular distribution of the GPCR or GRK may be determined. A difference may be monitored between (1) the distribution of the GPCR or GRK in the cell in the presence of the compound(s) and (2) the distribution of the GPCR or GRK in the cell in the absence of the compound(s). A difference may be correlated between (1) and (2) to the phosphorylation of the GPCR.

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