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US 7,405,053 B2Granted
Materials and methods relating to G-protein coupled receptor oligomers
AssigneeThe University Court of the University of Glasgow
InventorsGraeme Milligan, Dominic Behan
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Abstract
The invention provides materials and methods relating to G-protein coupled receptor (GPCR) oligomers. Complexes of two or more GPCRs associated with G-proteins are provided. Also provided are fusion proteins comprising a GPCR and a G-protein, nucleic acids, expression vectors and host cells. Methods of producing the complexes and fusion proteins of the invention are also provided.
Claims45
A biological reagent comprising a complex having
- (a) a first GPCR associated with a first G-protein wherein the first GPCR has a modified amino acid sequence compared to the wild-type GPCR sequence so as to render it non-functional with respect to the first G-protein;
- and;
- (b) a second GPCR associated with a second G-protein wherein the second G-protein is non-functional,
- wherein the wild-type form of the first GPCR is different to the second GPCR.
1 more element
A method of determining a first and second GPCR having affinity for each other such that they form a GPCR oligomer, said method comprising the steps of
- (a) producing or providing a first nucleic acid construct encoding a first GPCR and its associated G-protein as a fusion protein wherein the GPCR is mutated as compared to its wild-type GPCR so that it is non-functional with respect to its associated G-protein;
- (b) producing or providing a second nucleic acid construct encoding a second GPCR and its associated G-protein as a fusion protein wherein the G-protein is mutated as compared to its wild-type G-protein so that it is non-functional;
- (c) co-expressing said first and second nucleic acid constructs in a cell;
- and
- (d) determining the presence of a complex comprising said first and second GPCRs, wherein the wild type form of the first GPCR is different to the second GPCR.
2 more elements
A method for determining the presence of a new or altered ligand binding site resulting from GPCR oligomerisation, said method comprising the steps of
- a) contacting a compound with a first cell expressing a GPCR oligomer having (i) a first GPCR associated with a G-protein wherein the first GPCR is modified such that it is non-functional with respect to said G protein;
- and (ii) a second GPCR associated with a G-protein wherein the G-protein is modified so that it is non-functional;
- b) contacting said compound with a second cell expressing an unmodified first GPCR and/or contacting said compound with a second cell expressing an unmodified second GPCR;
- and
- c) comparing the effect of the compound on the first cell and the second cell to determine the presence of a new or modified ligand binding site created by the GPCR oligomer;
- wherein the wild-type form of the first GPCR is different to the second GPCR.
3 more elements
A method for determining a change in GPCR function as a result of forming a GPCR oligomer, said method comprising
- a) contacting a compound with a first cell expressing a GPCR oligomer having (i) a first GPCR associated with a G-protein wherein the first GPCR is modified such that it is non-functional with respect to said G protein;
- and (ii) a second GPCR associated with a G-protein wherein the G-protein is modified so that it is non-functional;
- b) contacting said compound with a second cell expressing an unmodified first GPCR and/or a second cell expressing an unmodified second GPCR;
- and
- (c) comparing the function of said GPCR oligomer with that of said unmodified first GPCR and/or with that of said second GPCR to determine a change in receptor function resulting from oligomerisation;
- wherein the wild-type form of the first GPCR is different to the second GPCR.
3 more elements
A method of determining an effect a compound has on a GPCR oligomer, comprising the steps of:
- a) contacting said compound with a first cell expressing a GPCR oligomer having (i) a first GPCR associated with a G-protein wherein the first GPCR has a modified amino acid sequence compared to the wild-type GPCR sequence so as to render it non-functional with respect to the first G-protein;
- and (ii) a second GPCR associated with a G-protein wherein the G-protein is modified so that it is non-functional;
- b) detecting the presence of a cellular signal resulting from contact between said compound and said GPCR oligomer;
- and
- c) determining an effect said compound has on the GPCR oligomer,
- wherein the wild-type form of the first GPCR is different to the second GPCR.
3 more elements
A method of identifying a compound having the ability to modulate binding between a GPCR oligomer and its ligand, said method comprising
- a) providing a cell expressing a GPCR oligomer comprising a (i) a first GPCR associated with a first G-protein wherein the GPCR is non-functional with respect to the G-protein;
- (ii) a second GPCR associated with a second G-protein wherein the second G-protein is non-functional;
- b) contacting said cell with a test compound in the presence of said ligand;
- and
- c) comparing the ability of said ligand to bind GPCR oligomer with the ability of said ligand to bind the GPCR oligomer under comparable conditions but in the absence of said compound;
- wherein the wild-type form of the first GPCR is different to the second GPCR.
3 more elements
A method for evaluating differential G-protein coupling comprising the steps of
- (a) providing a first cell expressing a GPCR oligomer comprising (i) a first GPCR associated with a first G protein wherein the GPCR is non-functional with respect to the G-protein;
- (ii) a second GPCR associated with a second G-protein wherein the second G-protein is non-functional;
- (b) providing a second cell expressing a GPCR oligomer comprising (i) said first GPCR associated with said first G-protein wherein said first GPCR is functional and said G-protein is non-functional;
- (ii) said second GPCR associated with said second G-protein wherein said second GPCR is non-functional with respect to G-protein;
- (c) providing a control cell expressing a monomer of said first GPCR associated with said first G-protein wherein both the GPCR and the G-protein are functional;
- (d) contacting said first, second and control cell with a compound capable of binding to the ligand binding site present on the first and/or the second GPCR;
- (e) repeating steps (a) to (d) with a different first and/or second G-protein;
- and
- (f) evaluating differential G-protein coupling;
- wherein the wild-type form of the first GPCR is different to the second GPCR.
7 more elements
A method for evaluating differential G-protein coupling for a GPCR comprising the steps of:
- (a) providing a first cell expressing a GPCR oligomer comprising (i) a first GPCR associated with a first G-protein wherein the GPCR is non-functional with respect to the G-protein;
- (ii) the second GPCR associated with a second G-protein wherein the second G-protein is non-functional;
- (b) providing a second cell expressing the second GPCR associated with the first G-protein wherein both the GPCR and the G-protein are functional;
- (c) contacting said first and second cells with a compound capable of binding to the ligand binding site present on the second GPCR;
- (d) repeating steps (a) to (c) one or more times with a different first G-protein;
- (e) optionally repeating steps (a) to (d) one or more times with a different first GPCR;
- and
- (f) evaluating differential G-protein coupling by the second GPCR;
- wherein the wild-type form of the first GPCR is different to the second GPCR.
6 more elements
Dependencies are inferred from the claim text.