Refolding of misfolded mutant GPCR: post-translational pharmacoperone action in vitro.
All reported GnRH receptor mutants (causing human hypogonadotropic hypogonadism) are misfolded proteins that cannot traffic to the plasma membrane. Pharmacoperones correct misfolding and rescue mutants, routing them to the plasma membrane where they regain function. Because pharmacoperones are often peptidomimetic antagonists, these must be removed for receptor function after rescue; in vivo this necessitates pulsatile pharmacoperone administration. As an antecedent to in vivo studies, we determined whether pharmacoperones need to be present at the time of synthesis or whether previously misfolded proteins could be refolded and rescued. Accordingly, we blocked either protein synthesis or intra-cellular transport. Biochemical and morphological studies using 12 mutants and 10 pharmacoperones representing three different chemical classes show that previously synthesized mutant proteins, retained by the quality control system (QCS), are rescued by pharmacoperones, showing that pharmacoperone administration in vivo likely need not consider whether the target protein is being synthesized at the time of drug administration.
Janovick
Jo Ann
JA
Divisions of Neuroscience and Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR 97006, USA.
Brothers
Shaun P
SP
Cornea
Anda
A
Bush
Eugene
E
Goulet
Mark T
MT
Ashton
Wallace T
WT
Sauer
Daryl R
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Haviv
Fortuna
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Greer
Jonathan
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D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D019556:COS Cells; D002522:Chlorocebus aethiops; D004353:Drug Evaluation, Preclinical; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007294:Inositol / Q000494:pharmacology; D018832:Molecular Chaperones / Q000494:pharmacology*; D018716:Molecular Mimicry; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011966:Receptors, LHRH / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D019086:Bridged Bicyclo Compounds, Heterocyclic; C461300:IN3 compound; D007211:Indoles; D018832:Molecular Chaperones; D050505:Mutant Proteins; D011725:Pyridines; D043562:Receptors, G-Protein-Coupled; D011966:Receptors, LHRH; D007294:Inositol