Phosphoinositide 3-kinase mediated inhibition of GPCRs
First Claim
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1. A method of screening compound(s) for modulating G protein-coupled receptor (GPCR) internalization, comprising the steps of:
- (a) providing a cell comprising molecules involved in GPCR internalization, wherein the molecules involved in GPCR internalization comprise β
-adrenergic receptor kinase 1 (β
ARK1), phosphoinositide 3-kinase (PI3K), GPCR, and arrestin, and wherein at least one of said molecules is detectably labeled;
(b) exposing the cell to the compound(s);
(c) identifying the location in the cell of the labeled molecule;
(d) comparing the location of the labeled molecule in the cell in the presence of the compound(s) to the location of the labeled molecule in the cell in the absence of the compound(s); and
(e) correlating a difference between (1) the location of the labeled molecule in the cell in the presence of the compound and (2) the location of the labeled molecule in the cell in the absence of the compound(s) to modulation of GPCR internalization.
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Abstract
The present invention relates to compounds that alter GPCR internalization and new methods for their identification. Compounds of this invention include modified phosphoinositide 3-kinase (PI3K), modified HEAT domain, modified β-adrenergic receptor kinase 1 (βARK1), as well as other peptides or small molecules that alter GPCR internalization. The present invention also includes the use of such compounds to treat GPCR-related diseases, such as cardiovascular disease, heart failure, asthma, nephrogenic diabetes insipidus, or hypertension.
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Citations
58 Claims
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1. A method of screening compound(s) for modulating G protein-coupled receptor (GPCR) internalization, comprising the steps of:
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(a) providing a cell comprising molecules involved in GPCR internalization, wherein the molecules involved in GPCR internalization comprise β
-adrenergic receptor kinase 1 (β
ARK1), phosphoinositide 3-kinase (PI3K), GPCR, and arrestin, and wherein at least one of said molecules is detectably labeled;
(b) exposing the cell to the compound(s);
(c) identifying the location in the cell of the labeled molecule;
(d) comparing the location of the labeled molecule in the cell in the presence of the compound(s) to the location of the labeled molecule in the cell in the absence of the compound(s); and
(e) correlating a difference between (1) the location of the labeled molecule in the cell in the presence of the compound and (2) the location of the labeled molecule in the cell in the absence of the compound(s) to modulation of GPCR internalization. - View Dependent Claims (47, 54, 55, 56)
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2-23. -23. (canceled)
- 24. A modified PI3K, wherein GPCR desensitization is altered when said modified PI3K is expressed in a cell.
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38. A modified β
- ARK1 which lacks the ability to bind PI3K.
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45-46. -46. (canceled)
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48. A kit for detecting a β
- ARK1/PI3K complex in a biological sample comprising an antibody which recognizes and binds to the β
ARK1/PI3K complex and reagents which detect the antibody that binds to the β
ARK1/PI3K complex.
- ARK1/PI3K complex in a biological sample comprising an antibody which recognizes and binds to the β
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49. An isolated immunoglobulin which binds to a β
- ARK1/PI3K complex.
- View Dependent Claims (50)
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51. (canceled)
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52. A method of altering GPCR internalization, comprising providing an effective amount of LY294002 or wortmannin.
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53. (canceled)
Specification