Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor

Biology of Reproduction
Joseph E MazurkiewiczJames A Dias

Abstract

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma ...Continue Reading

References

May 1, 1992·Molecular and Cellular Endocrinology·R S Weiner, J A Dias
Apr 1, 1995·Biophysical Journal·D B ZimetJ R Abney
Jul 2, 1999·Biophysical Journal·Y ChenE Gratton
Oct 21, 1999·Methods : a Companion to Methods in Enzymology·Y ChenE Gratton
Jan 4, 2001·Journal of Reproductive Immunology·B Lindau-ShepardJ A Dias
Feb 23, 2002·The Journal of Biological Chemistry·ChangWoo LeeTae H Ji
Jan 25, 2003·Methods : a Companion to Methods in Enzymology·Masilamani ElangovanAmmasi Periasamy
Jun 28, 2003·Biophysical Journal·Horst WallrabeMargarida Barroso
Apr 9, 2004·Proceedings of the National Academy of Sciences of the United States of America·S J BriddonS J Hill
Jun 24, 2004·Molecular Pharmacology·Graeme Milligan
Jul 22, 2005·Endocrine·James A DiasRaghad Atari
Mar 16, 2006·Microscopy Research and Technique·Horst WallrabeMargarida Barroso
Jun 8, 2006·Journal of Assisted Reproduction and Genetics·Takehiro SerikawaKenichi Tanaka
Nov 9, 2007·Proceedings of the National Academy of Sciences of the United States of America·Gyun Jee SongPatricia M Hinkle
Jan 19, 2010·Proceedings of the National Academy of Sciences of the United States of America·Adolfo Rivero-MüllerIlpo Huhtaniemi
Nov 17, 2010·Pharmacological Reviews·Nicola J Smith, Graeme Milligan
May 25, 2011·Methods in Molecular Biology·Stephen J BriddonStephen J Hill
Jun 22, 2011·The Journal of Biological Chemistry·Amber L Wolf-RingwallB George Barisas
Jul 18, 2012·Proceedings of the National Academy of Sciences of the United States of America·Xuliang JiangXiaolin He
Mar 26, 2013·The Biochemical Journal·Suparna PatowaryValerică Raicu

❮ Previous
Next ❯

Citations

Oct 7, 2015·Frontiers in Endocrinology·Selvaraj G NatarajaStephen S Palmer
Aug 4, 2015·Frontiers in Endocrinology·Antara A Banerjee, Smita D Mahale
May 7, 2016·Journal of Molecular Endocrinology·Gunnar KleinauHeike Biebermann
May 26, 2016·Reproduction : the Official Journal of the Society for the Study of Fertility·Xing DuQifa Li
Jul 18, 2016·Molecular and Cellular Endocrinology·Mohammed Akli AyoubEric Reiter
Oct 19, 2016·Molecular Pharmacology·Alfredo Ulloa-Aguirre, Teresa Zariñán
Jan 25, 2017·Molecular and Cellular Endocrinology·K C Jonas, A C Hanyaloglu
Jul 10, 2018·Endocrinology·Alfredo Ulloa-AguirrePascale Crépieux
Dec 29, 2019·Biology of Reproduction·Gillian P Johnson, Kim Carol Jonas
May 26, 2018·Endocrinology·Selvaraj NatarajaStephen Palmer
Mar 6, 2019·Frontiers in Endocrinology·Diego GuidolinLuigi F Agnati
Dec 18, 2018·Frontiers in Endocrinology·Alfredo Ulloa-AguirreJames A Dias
Jan 9, 2019·Frontiers in Endocrinology·Kamila SzymańskaAdolfo Rivero-Müller
Oct 30, 2020·The Journal of Clinical Endocrinology and Metabolism·Teresa ZariñánAlfredo Ulloa-Aguirre
Oct 22, 2020·Molecular Human Reproduction·Livio CasariniManuela Simoni

❮ Previous
Next ❯

Methods Mentioned

BETA
FRET
fluorescence correlation spectroscopy
PCR
Transfection
FCS
Fluorescence

Software Mentioned

Precision FRET Analysis ( PFRET )
ImageJ
Zeiss Aim
AIM
Excel
PFRET

Related Concepts

Related Feeds