Mutational heterogeneity represents a significant barrier to development of therapies for many inherited diseases. In several instances, the outcome of the mutation is fairly easy to predict. For example, the presence of a premature stop codon that leads to a truncated receptor after the first TM domain certainly leads to a protein that, even if inserted in the plasma membrane, is unlikely to bind ligand or transduce signal. In contrast, missense mutations that result in the substitution of a single amino acid in the encoded protein theoretically could have three major effects: (1) mutations that cause loss of stability or misfolding of the protein, and thus interfere with the normal targeting and insertion of the receptor into the plasma membrane; (2) mutations that affect residues involved in the formation of the binding site, and thus prevent binding of the ligand or drastically reduce the receptor' s affinity for it; and (3) mutations that affect domains involved in transducing the signal from the receptor to signaling partners, thus leading to a receptor that can bind the ligand but cannot propagate the signal to downstream effectors. As approximately two-thirds of the mutations in GPCRs that cause human disease are missense, it is important to determine the functional consequences of these more subtle mutations. In recent years, this has been accomplished by expressing the DNA encoding the missense mutated receptors in defined heterolo-gous cellular systems that have little to no endogenous receptor. The ability of the mutant receptors to insert in the plasma membrane, bind agonist, interact with G proteins and stimulate downstream effectors, and undergo regulation can then be compared with the characteristics of the wild-type receptors expressed in the same system. This approach allows one to determine which aspects of receptor function are impaired by specific mutations. As mentioned earlier, over 40% of the missense mutations, and small in-frame insertions and deletions are predicted to result in less stable or misfolded trafficking-defec-tive GPCRs (for review, see References 2, 3) but do not necessarily affect other receptor functions, such as ligand binding and signal transduction. Mutated GPCRs of this type are the ones most likely to be responsive to a pharmacological chaperone that specifically binds to and stabilizes the receptor, promoting folding and efficient trafficking to the plasma membrane, where the otherwise functional mutant receptor can carry out its physiological function. Missense mutant GPCRs that are unlikely to respond to a pharmacological chaperone include those that fold and traffic normally, but are deficient in ligand binding or coupling, those with severe folding deficiencies that cannot be effectively stabilized by a small molecule, or those with gross alterations in structure. In diseases caused by a large number of different missense mutations in different domains of a GPCR, it is important to clearly understand the nature of the molecular defect in vitro. Efforts should then be focused on those mutant forms that are trafficking defective and functionally rescuable, and hence define the potentially responsive patient population, prior to testing a pharmacological chaperone in a clinical setting.

Determining the mutant forms of a GPCR that are most likely amenable to pharmacological chaperone therapy is relatively straightforward as responsive mutant forms will show increased cell surface expression, ligand binding, and/or intracellular signaling after incubation with the chaperone in cell culture. However, a period of compound washout after exposure is required prior to measuring ligand binding or intracellular signaling as many of the pharmacological chaperones for GPCRs that have been described are competitive or reversible antagonists (see below). In contrast, increases in cell surface expression can be measured even in the presence of the pharmacological chaperone by using epitope-tagged receptors. As described above, 11 of 13 missense mutant forms of GnRHR with confirmed or suspected trafficking defects were responsive to IN3, as shown by increased ligand binding and intracellular signaling after incubation [154-156] . The responses of mutant forms of GnRHRs to IN3 were of variable magnitude, with the level of functional rescue ranging from fully restored to near wild- type levels to nonre-sponsive, meaning that no increases in ligand binding or intracellular signaling were detected after incubation with the pharmacological chaperone. Similarly, various levels of both cell surface expression and function for 25 mutant forms of the V2R that are found in NDI were responsive to pharmacological chaperones (for review, see References 58, 62).

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