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Rescue of misrouted GnRHR mutants reveals its constitutive activity

Jo Ann Janovick1, Irina D Pogozheva, Henry I Mosberg

  • 1Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, Oregon 97006-3448, USA.

Molecular Endocrinology (Baltimore, Md.)
|May 19, 2012
PubMed

Insights

Researchers identified novel human GnRH receptor (GnRHR) mutants exhibiting constitutive activity (CA). These mutants, previously retained by cellular quality control, were rescued to the plasma membrane, revealing significant CA levels comparable to agonist activation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • G protein-coupled receptors (GPCRs) are crucial in physiology, with mutations causing hereditary diseases.
  • Constitutive activity (CA) in GPCR mutants means they activate signaling without agonist binding.
  • The GnRH receptor (GnRHR) was previously lacking identified CA mutants due to cellular retention.

Purpose of the Study:

  • To identify and characterize novel constitutively active human GnRH receptor (hGnRHR) mutants.
  • To investigate methods for rescuing misfolded CA mutants from endoplasmic reticulum retention.
  • To analyze the conformational basis of CA in hGnRHR mutants.

Main Methods:

  • Site-directed mutagenesis to create hGnRHR mutants (F(272)K, F(272)Q, Y(284)F, C(279)A, C(279)S).
  • Pharmacoperone treatment and K(191) deletion to rescue mutants for plasma membrane trafficking.
  • Confocal microscopy to visualize receptor localization.
  • Inositol phosphate production assays to measure receptor activity.
  • Computer modeling to evaluate mutant conformations.

Main Results:

  • Several hGnRHR mutants, particularly F(272)K and F(272)Q, demonstrated significant CA after rescue.
  • Rescue strategies successfully relocated mutants from the endoplasmic reticulum to the plasma membrane.
  • Rescued F(272)K and F(272)Q mutants exhibited CA levels comparable to agonist-stimulated activation.
  • Computer modeling provided insights into the conformations associated with CA.

Conclusions:

  • The study successfully identified and characterized novel constitutively active hGnRHR mutants.
  • Pharmacoperones and genetic modifications can overcome cellular retention of misfolded CA mutants.
  • This approach offers a potential strategy for discovering CA in other GPCR systems.
  • Understanding the conformational basis of CA is crucial for receptor function studies.

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