Systems for sensitive detection of G-protein coupled receptor and orphan receptor function using reporter enzyme mutant complementation

A method of assessing the effect of a test condition on G-protein-coupled receptor (GPCR) pathway activity, comprising:

• a) providing a cell that expresses a GPCR as a fusion protein to one mutant form of reporter enzyme and an interacting protein partner as a fusion to another mutant form of enzyme;
• wherein said GPCR fusion protein is modified to include one or more sets of serine/threonine clusters, wherein said one or more sets of serine/threonine clusters enhance binding of said GPCR to arrestin, wherein said enhanced binding between said GPCR and said arrestin increases sensitivity of detection of said effect of said test condition;
• b) exposing the cell to a ligand for said GPCR under said test condition;
• and
• c) monitoring activation of said GPCR by complementation of said reporter enzyme;
• wherein increased reporter enzyme activity in the cell compared to that which occurs in the absence of said test condition indicates increased GPCR interaction with said interacting protein partner compared to that which occurs in the absence of said test condition, and decreased reporter enzyme activity in the cell compared to that which occurs in the absence of said test condition indicates decreased GPCR interaction with interacting protein partner compared to that which occurs in the absence of said test condition.

A method of assessing the effect of a test condition on G-protein-coupled receptor (GPCR) pathway activity, comprising:

• a) providing a cell that expresses a GPCR as a fusion protein to one mutant form of reporter enzyme and an interacting protein partner as a fusion to another mutant form of enzyme,
• wherein said cell also expresses an arrestin, wherein said arrestin is modified by introducing a point mutation in a phosphorylation-recognition domain to remove a requirement for phosphorylation of said GPCR for arrestin binding to permit binding of said arrestin to said GPCR in said cell regardless of whether said GPCR is phosphorylated,
• b) exposing the cell to a ligand for said GPCR under said test condition;
• and
• c) monitoring activation of said GPCR by complementation of said reporter enzyme;
• wherein increased reporter enzyme activity in the cell compared to that which occurs in the absence of said test condition indicates increased GPCR interaction with its interacting protein partner compared to that which occurs in the absence of said test condition, and decreased reporter enzyme activity in the cell compared to that which occurs in the absence of said test condition indicates decreased GPCR interaction with its interacting protein partner compared to that which occurs in the absence of said test condition.

A method of assessing the effect of a test condition on G-protein-coupled receptor (GPCR) pathway activity, comprising:

• a) providing a cell that expresses a GPCR as a fusion protein to one mutant form of reporter enzyme and an interacting protein partner as a fusion to another mutant form of enzyme;
• wherein said GPCR fusion protein is modified to include one or more sets of serine/threonine clusters, said one or more serine/threonine clusters defined as serine or threonine residues occupying three consecutive or three out of four positions in a carboxyl-termini of said GPCR, wherein said one or more sets of serine/threonine clusters enhance binding of said GPCR to arrestin, wherein said enhanced binding between said GPCR and said arrestin increases sensitivity of detection of said effect of said test condition;
• b) exposing the cell to a ligand for said GPCR under said test condition;
• and
• c) monitoring activation of said GPCR by complementation of said reporter enzyme;
• wherein increased reporter enzyme activity in the cell compared to that which occurs in the absence of said test condition indicates increased GPCR interaction with said interacting protein partner compared to that which occurs in the absence of said test condition, and decreased reporter enzyme activity in the cell compared to that which occurs in the absence of said test condition indicates decreased GPCR interaction with interacting protein partner compared to that which occurs in the absence of said test condition.