Trafficking and quality control of the gonadotropin releasing hormone receptor in health and disease

Abstract

In order to serve as enzymes, receptors and ion channels, proteins require structural precision. This is monitored by a cellular quality control system (QCS) that rejects misfolded proteins and thereby protects the cell against aberrant activity. Misfolding can result in protein molecules that retain intrinsic function, yet become misrouted within the cell; these cease to perform normally and result in disease. A therapeutic opportunity exists to correct misrouting and rescue mutants using "pharmacoperones" (small molecular folding templates, often peptidomimetics, which promote correct folding and rescue) thereby restoring function and potentially curing the underlying disease. Because of its small size, the GnRH (gonadotropin-releasing hormone) receptor (GnRHR) is an excellent model for GPCR (G protein-coupled receptor) and has allowed elucidation of the precise biochemical mechanism of pharmacoperone action necessary for rational design of new therapeutic agents. This review summarizes what has been learned about the structural requirements of the GnRHR that govern its interaction with the QCS and now presents the potential for the rational design of pharmacoperones. Because of the role of protein processing, this approach is likely to be applicable to other GCPCs and other proteins in general.

Figure 1. Model of the human GnRH receptor showing residues of interest referred to in the text

Circles represent amino acids; those colored green form a motif of four non-contiguous residues that are required in the human GnRHR for Lys¹⁹¹ (red) to destabilize the formation of the Cys¹⁴-Cys²⁰⁰ (Cys¹⁹⁹ in the rat or mouse) bridge (gray). Circles colored orange represent amino acids for the two naturally occurring mutants that cannot be rescued by known pharmacoperones. Thermodynamically unfavored modifications at these sites (both Ser → Arg) cause misfolding due to misalignment of the Cys residues that normally form the Cys¹⁴-Cys²⁰⁰ bridge, as described in the text. The site of pharmacoperone (shown as “P” in the white box) action (bridging residues Asp⁹⁸ to Lys¹²¹) is shown, as is the naturally occurring Glu⁹⁰-Lys¹²¹ salt bridge. This portion of the hGnRHR is also shown in the enlarged detail of transmembrane segments 2 and 3. The mammalian GnRHR lacks the long cytoplasmic tail (COOH terminal) that is typical of this super-family and present in piscine, reptilian and avian GnRHRs.

Figure 2. Effect of mutation of residue Asp⁹⁸ to Ala, Lys, or Asn on rescue with two classes of pharmacoperones

Total IP production was measured in response to a saturating concentration of Buserelin (10⁻⁷ M). Data are expressed as percent of WT without pharmacoperone (panel A) or after preincubation (rescue) with A177775 (panel B) or TAK-013 (panel C). Cells were transfected with 95 ng of WT or mutant cDNA. Mutants Ser¹⁶⁸Arg and Ser²¹⁷Arg were used as controls, since these are grossly misfolded and cannot be rescued from ER retention with pharmacoperone. Mutant Glu⁹⁰Lys was included as a positive control for rescue with pharmacoperone. Inset illustration shows the dose response curves (log scale) for each class of pharmacoperone rescue. Averages and SEMs of at least 3 independent experiments performed in replicates of 6 are shown. The dose-response for rescue and chemical structures are shown to the right of graphics B–C.

Figure 3. Dominant-negative effect of co-transfecting cells with 5 ng WT and 95 ng mutant cDNA (1:19) on control (panel A) or pharmacoperone rescue with A177775 (panel B) or TAK-013 (panel C) as described in the text

Averages and SEMs of at least 3 independent experiments performed in replicates of 6 are shown.

Figure 4. Effect of mutation of residue Lys¹²¹ to Ala, Asp, Glu, Gly, Asn, Gln or Arg on rescue with two classes of pharmacoperones

Cells were transiently transfected with 25 ng of WT hGnRHR or mutant cDNA in which residue Lys¹²¹ was replaced by Ala, Asp, Glu, Gly, Asn, Gln or Arg. Cells were treated with or without pharmacoperone TAK-013 or A177775 and IP response was measured to a saturating dose of Buserelin (10⁻⁷ M). Empty vector (pcDNA3.1) was run as a control and was typically 175 ± 20 cpm. Averages and SEMs were calculated from at least 3 independent experiments performed in replicates of 6.

Figure 5. Displacement of 125I-Buserelin binding to the GnRHR by pharmacoperones TAK-013 or A177775

Cells were plated, transfected with the indicated vector and incubated in 1.25 × 10⁵ cpm/ml of [¹²⁵I]-Buserelin in the presence of the indicated concentrations of pharmacoperones A177775 or TAK-013. Binding was determined as described in Janovick et al., 2009.