G protein coupled receptor agonists and antagonists and methods of activating and inhibiting g protein coupled receptors using the same

Abstract

The invention relates generally to G protein coupled receptors and in particular to agonists and antagonists of G protein receptors and methods of using the same.

1 - 22 . (canceled) 
     
     
         23 . A method for identifying an agent that modulates the activity or function of a G protein coupled receptor (GPCR), the method comprising:
 a. providing a cell comprising said GPCR, or having a property or function ascribable to said GPCR;   b. contacting said cell with a polypeptide, said polypeptide comprising:
 i. a first domain comprising an intracellular loop, or a fragment thereof, of said GPCR, wherein said fragment comprises at least 3 contiguous amino acid residues of the intracellular loop; and 
 ii. a second domain, attached to the first domain, wherein said second domain comprises a cell-penetrating, membrane-tethering hydrophobic moiety comprising: a lipid, cholesterol, a phospholipid, a steroid, a sphingosine, a ceramide, octyl-glycine, 2-cyclohexylalanine, or benzolylphenylalanine; 
 wherein said polypeptide is an agonist or antagonist of said GPCR; and 
   c. contacting said cell with a candidate agent;   d. determining whether said candidate agent alters the activity or function of said GPCR;   wherein, an alternation in the activity or function of said GPCR in the presence of said candidate agent, a as compared to a control in which a cell is not exposed said candidate agent, is indicative that said agent modulates the activity or function of said GPCR.   
     
     
         24 . The method of  claim 23 , wherein said polypeptide is an agonist of its cognate GPCR. 
     
     
         25 . The method of  claim 24 , wherein a decrease in the activity or function of said GPCR in the presence of said candidate agent, a as compared to a control in which a cell is not exposed said candidate agent, is indicative that said agent is an antagonist of said GPCR. 
     
     
         26 . The method of  claim 23 , wherein said activity or function of the GPCR is determined by a second messenger assay. 
     
     
         27 . The method of  claim 26 , wherein said second messenger is intracellular Ca 2+ , diacylglycerol, or IP 3 . 
     
     
         28 . The method of  claim 23 , wherein said GPCR is a Class A or Class B GPCR. 
     
     
         29 . The method of  claim 23 , wherein said GPCR is a mammalian GPCR. 
     
     
         30 . The method of  claim 23 , wherein the second domain comprises a moiety that is a tridecanoyl (C13), myristoyl (C14), pentadecanoyl (C15), palmitoyl (C16), phytanoyl (methyl substituted C16), heptadecanoyl (C17), stearoyl (C18), nonadecanoyl (C19), arachidoyl (C20), heniecosanoyl (C21), behenoyl (C22), trucisanoyl (C23), or lignoceroyl (C24) moiety. 
     
     
         31 . The method of  claim 23 , wherein the second domain comprises a moiety that is a palmitoyl (C16), pentadecanoyl (C15) or myristoyl (C14) moiety. 
     
     
         32 . A method for identifying an agent that modulates the activity or function of a G protein coupled receptor (GPCR), the method comprising:
 a. providing a composition comprising said GPCR in membrane-bound form;   b. contacting said membrane-bound GPCR with a polypeptide, said polypeptide comprising:
 i. a first domain comprising an intracellular loop, or a fragment thereof, of said GPCR, wherein said fragment comprises at least 3 contiguous amino acid residues of the intracellular loop; and 
 ii. a second domain, attached to the first domain, wherein said second domain comprises a membrane-tethering hydrophobic moiety comprising: a lipid, cholesterol, a phospholipid, a steroid, a sphingosine, a ceramide, octyl-glycine, 2-cyclohexylalanine, or benzolylphenylalanine; 
 wherein said polypeptide is an agonist or antagonist of said GPCR; and 
   c. contacting said membrane-bound GPCR with a candidate agent;   d. determining whether said candidate agent alters the activity or function of said GPCR;   wherein, an alternation in the activity or function of said GPCR in the presence of said candidate agent, a as compared to a control in which said GPCR is not exposed said candidate agent, is indicative that said agent modulates the activity or function of said GPCR.   
     
     
         33 . The method of  claim 32 , wherein said polypeptide is an agonist of its cognate GPCR. 
     
     
         34 . The method of  claim 33 , wherein a decrease in the activity or function of said GPCR in the presence of said candidate agent, a as compared to a control in which a cell is not exposed said candidate agent, is indicative that said agent is an antagonist of said GPCR. 
     
     
         35 . The method of  claim 32 , wherein said activity or function of the GPCR is determined by a second messenger assay. 
     
     
         36 . The method of  claim 35 , wherein said second messenger is intracellular Ca 2+ , diacylglycerol, or IP 3 . 
     
     
         37 . The method of  claim 32 , wherein said GPCR is a Class A or Class B GPCR. 
     
     
         38 . The method of  claim 32 , wherein said GPCR is a mammalian GPCR. 
     
     
         39 . The method of  claim 32 , wherein the second domain comprises a moiety that is a tridecanoyl (C13), myristoyl (C14), pentadecanoyl (C15), palmitoyl (C16), phytanoyl (methyl substituted C16), heptadecanoyl (C17), stearoyl (C18), nonadecanoyl (C19), arachidoyl (C20), heniecosanoyl (C21), behenoyl (C22), trucisanoyl (C23), or lignoceroyl (C24) moiety. 
     
     
         40 . The method of  claim 32 , wherein the second domain comprises a moiety that is a palmitoyl (C16), pentadecanoyl (C15) or myristoyl (C14) moiety.