Pharmacochaperones Post-translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-type and Mutant Vasopressin V2 Receptors

Abstract

Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V 2 receptor GFP fusion protein (mV 2 R⅐GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC 50 values similar to their K D values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V 2 Rs (hV 2 Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV 2 R.Water homeostasis in mammals is regulated through arginine-vasopressin (AVP), 1 acting through the vasopressin V 2 receptor (V 2 R) expressed in the renal collecting duct (1). In Xlinked nephrogenic diabetes insipidus (NDI), the kidney shows a resistance to the action of AVP, caused by inactivating mutations of the human V 2 R (hV 2 R) gene (2). More than 150 different mutations have been described (for review, see Ref.3), 50% of which are missense mutations resulting in the substitution of a single amino acid. Most of the hV 2 R mutants with a single amino acid exchange are retained within the ER and not transported to the cell surface (for review, see Ref.3). Most likely, the amino acid exchanges result in improper folding of the mutant hV 2 Rs and subsequently prolonged association with molecular chaperones. For example, for the NDI mutant hR337X, a prolonged association with the ER-chaperone calnexin has been observed (4). Chaperone association prevents the aggregation of misfolded proteins, but also inhibits the exit of improperly folded proteins from the ER until correct folding is established.Recently, it has been found that membrane-permeable antagonists not only inhibit receptor activation, but also promote cell surface expression of misfolded, ER-retained G proteincoupled receptors (GPCRs). This concept represents an intriguing new approach for the therapy of congenital diseases caused by mutations in genes encoding GPCRs. For the ER-retained rhodopsin mutant P23H (a frequent cause of autosomal-dominant retinitis pigmentosa), it has been shown in vitro that the inverse agonist 9-cis-retinal or the non-hydrolyzable inverse agonist 11-cis-7-ring-retinal promoted cell surface expression (5,6). Restoration of cell surface expression by antagonists or inverse agonists has also been...

Intracellular activation of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus by nonpeptide agonists

“…4)(10-12, 17). The absence of translocation of V2R mutants to the cell surface is further supported by the fact that cAMP responses are obtained within 10 min following agonists addition, whereas it takes V2R antagonists 4-8 h to insert V2R mutants in the plasma membrane (11,23). Our data are different from those of Petaja-Repo et al (24), who found that nonpeptide agonists were able to stabilize the expression and maturation of ER-retained wild-type ␦-opioid receptor (DOR).…”

Refolding of misfolded mutant GPCR: Post-translational pharmacoperone action in vitro

“…The present study is a needed antecedent to selection of the appropriate time for pulsatile administration of pharmacoperones in vivo and suggests that, at least in the case of GnRH receptor mutants with three chemical classes of pharmacoperones, the drug need not be present at the time of synthesis, as appears to be the case for other vasopressin receptor subtypes (Hawtin, 2006;Morello et al, 2000;Wuller et al, 2004). The present model suggests that pharmacoperones act post-translationally to aid in receptor folding.…”

Luteinizing Hormone Receptor Ectodomain Splice Variant Misroutes the Full-Length Receptor into a Subcompartment of the Endoplasmic Reticulum

“…Recent evidence suggests that the ER quality control does not only target permanently misfolded proteins to ERAD but may also dispose some folding competent ones, possibly due to their slow folding kinetics. This applies also to several polytopic membrane proteins, including G protein-coupled receptors (GPCRs; Petäjä-Repo et al, 2000;Imai et al, 2001;Lu et al, 2003;Wü ller et al, 2004;Pietilä et al, 2005). Thus, export from the ER is the limiting step in their expression and probably provides a mean to regulate the number of functional receptors at the cell surface.…”