STRATEGY FOR CLONING AND EXPRESSING THE EXTRACELLULAR DOMAINS OF RECEPTORS AS SOLUBLE PROTEINS
First Claim
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1. A soluble polypeptide that retains three dimensional conformation of a native G protein-coupled receptor (GPCR) protein comprising:
- at least two extracellular domains of a GPCR protein, linked in tandem by short inter-domain linkers to form a soluble polypeptide that retains three dimensional conformation of a native GPCR corresponding to the extracellular domains of the GPCR protein, wherein the soluble polypeptide is capable of binding a ligand or functionally equivalent analog thereof for the native GPCR protein.
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Abstract
The present invention relates generally to soluble G protein-coupled receptor constructs. More specifically, the invention relates to soluble chemokine receptors, and soluble HIV co-receptors in particular. The invention is generally useful for designing and constructing soluble GPCR, which may be used to identify binding molecules. The invention also relates to methods of treating and/or preventing a disease or disorder associated with impaired function of such a receptor. The invention thus provides compositions and methods for therapeutic applications, such as vaccine.
25 Citations
84 Claims
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1. A soluble polypeptide that retains three dimensional conformation of a native G protein-coupled receptor (GPCR) protein comprising:
at least two extracellular domains of a GPCR protein, linked in tandem by short inter-domain linkers to form a soluble polypeptide that retains three dimensional conformation of a native GPCR corresponding to the extracellular domains of the GPCR protein, wherein the soluble polypeptide is capable of binding a ligand or functionally equivalent analog thereof for the native GPCR protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26-38. -38. (canceled)
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39. A method for identifying a molecule that binds to a G protein-coupled receptor (GPCR) protein of native conformation, the method comprising:
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contacting a sample containing at least one test molecule with a soluble polypeptide that retains three dimensional conformation of a native GPCR protein, comprising extracellular domains of a GPCR protein, linked in tandem by short inter-domain linkers, wherein the soluble polypeptide retains three dimensional conformation of the native GPCR, and identifying the molecule that binds to the polypeptide.
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40-67. -67. (canceled)
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68. A method of inhibiting ligand-dependent receptor stimulation of a G protein-coupled receptor (GPCR), the method comprising:
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contacting a cell expressing the GPCR on the cell surface with a soluble polypeptide that retains three dimensional conformation of a native GPCR protein comprising; at least two extracellular domains of a GPCR protein, linked in tandem by short inter-domain linkers to form a soluble polypeptide that retains three dimensional conformation of a native GPCR corresponding to the extracellular domains of the GPCR protein, wherein the soluble polypeptide is capable of binding a ligand or functionally equivalent analog thereof for the native GPCR protein in an amount effective for inhibiting ligand-dependent receptor stimulation of a GPCR.
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69-71. -71. (canceled)
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72. A method of treating an HIV infection, the method comprising:
administering to a subject in need of such treatment a composition comprising a soluble polypeptide that retains native three-dimensional conformation of extracellular portions of an HIV co-receptor, comprising at least part of an N-terminus, an ECL1 domain, an ECL2 domain and an ECL3 domain of the HIV co-receptor, linked in tandem by inter-domain PGGS linkers and disulfide bonds, in a pharmacologically effective amount to treat HIV infection.
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73-81. -81. (canceled)
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82. A method of treating a disease or disorder caused by a G protein-coupled receptor (GPCR) mutation, wherein the GPCR mutation is associated with altered basal activity, the method comprising:
administering to a subject in need of such treatment a composition comprising a soluble polypeptide that retains native three-dimensional conformation of extracellular portions of a GPCR, comprising at least part of an N-terminus, an ECL1 domain, an ECL2 domain and an ECL3 domain of the GPCR, linked in tandem by inter-domain linkers, wherein at least one of the inter-domain linkers comprises a PGGS linker, and disulfide bonds, in an amount effective to treat the disease or disorder caused by a GPCR mutation. - View Dependent Claims (83)
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84-102. -102. (canceled)
Specification