Pharmacological Chaperones Correct Misfolded GPCRs and Rescue Function: Protein Trafficking as a Therapeutic Target - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 266

other structural features are required for the effects of this residue on the human
receptor (Janovick et al. 2006 ). In fact, a number of motifs in multiple domains of
the hGnRHR, that control the destabilizing influence of K 191 on the C 14 -C 200 bridge
and limit its PM expression, have been identified (Janovick et al. 2006 ; Ulloa-
Aguirre et al. 2006 ). These data unveiled a novel and underappreciated mechanism
for posttranslational control of a GPCR by altering its interaction with the ER QCS
and provided a biochemical explanation for the pathogenesis of disease-causing
mutations of this receptor (Conn et al. 2006b ) .
As shown in Fig. 14.1b, c , 21 inactivating mutations (including two deletions of
large sequences) in the hGnRHR have been described as a cause of complete or
partial HH (Beranova et al. 2001 ; Conn and Ulloa-Aguirre 2010 ; Ulloa-Aguirre
et al. 2004b ). Seven homozygous and twelve heterozygous combinations of hGn-
RHR mutants are expressed by individuals exhibiting either partial or complete
forms of HH (Conn and Ulloa-Aguirre 2010 ). Expression of these hGnRHR mutants
in in vitro heterologous systems resulted in cells that bind poorly to GnRH agonists
and, consequently, that showed no, or minimal, response to GnRH stimulation by
effector activation. At first sight, these observations suggested that such mutations
were associated with alterations in agonist binding, receptor activation or interac-
tion with coupled effectors. However, the majority (~90%) of GnRHR mutants,
whose function has been examined to date (19 mutants), are trafficking-defective
receptors as disclosed by mutational studies and/or response to pharmacoperones
(Ulloa-Aguirre et al. 2004b ). Because reproductive failure is not life-threatening, it
is likely that many partial forms of HH go undiagnosed and, individual mutants, if
severe in phenotype, are not inherited to progeny. Such misfolded mutants fre-
quently exhibit a change in residue charge (e.g. the E 90 K hGnRHR mutant), or gain
(e.g. the Y 108 C mutant) or loss of either cysteine residues (necessary to form bridges
associated with the third-order structure of proteins, e.g. the C 200 Y mutant) or a
proline (an amino acid residue frequently associated with a force turn in the protein
sequence, e.g. the P 320 L mutant) (Fig. 14.1b ). These structural features, as well as
motifs involved in the control of the destabilizing influence of K 191 on the C 14 -C 200
bridge formation, may explain some of the mechanisms whereby mutations in the
hGnRHR lead to defective intracellular traffic. For some hGnRHR mutants causing
HH, we have elucidated these mechanisms:
(a) The naturally occurring mutations at positions 168 (S 168 K) and 217 (S 217 K) in
the TMs 4 and 5, respectively, represent thermodynamically unfavored substi-
tutions that “twist” the corresponding a-helices; this moves the eL2 away from
the NH 2 -terminal domain and thus prevents formation of the critical C 14 -C 200
bridge. These mutant hGnRHRs never pass the ER QCS and are completely
recalcitrant to either rescuing approach (Janovick et al. 2003a, 2006 ; Leanos-
Miranda et al. 2002 ; Ulloa-Aguirre et al. 2006 ) .
(b) In the misfolded E 90 K mutant (located at the TM2), the dramatic shift in charge
prevented the formation of an E 90 -K 121 salt bridge required to stabilize the interac-
tion between the TMs 2 and 3 (Fig. 14.1d ) that is apparently required to pass the