Use of pharmacoperones to reveal GPCR structural changes associated with constitutive activation and trafficking

The gonadotropin-releasing hormone (GnRH) receptor (GnRHR), because of its small size among G-protein-coupled receptors (GPCRs), is amenable to facile preparation of mutants. This receptor is used in our laboratory as a structural model for this super-family of protein receptors and has helped us understand the requirements for proper trafficking. We have demonstrated that pharmacoperones ("pharmacological chaperones"), small target-specific drugs that diffuse into cells, are capable of rescuing misfolded and misrouted GnRHR mutants and restoring them to function. By rescuing these proteins, these drugs enable the plasma membrane expression of such mutants in living cells and allow examination of mutants that would otherwise be retained in the endoplasmic reticulum and would not be available for ligand binding and signal transduction. As an example of the efficacy of this method, we have shown that mutant E⁹⁰K, which breaks a salt bridge (E⁹⁰-K¹²¹) normally found in the GnRHR, results in constitutive activity when rescued by pharmacoperones. A second method of rescue, involving a mutation that increases the expression of GnRHRs, is shown to have a similar effect. Normally, in the absence of rescue by either of these methods, this mutant, associated with human hypogonadotropic hypogonadism, is misrouted and this constitutive activity has gone unrecognized. This observation [Janovick, J. A., and Conn, P. M. (2010). Salt bridge integrates GPCR activation with protein trafficking. Proc. Natl. Acad. Sci. USA107, 4454-4458.] showed that the cell normally recognizes this protein as defective and prevents its routing to the plasma membrane.