Barry University, Florida, USA1 Hormone Research Center, R. of Korea2

1
Abstract 371 It is now established that two or
more forms of gonadotropin releasing hormone
(GnRH) are expressed in the brain of several
vertebrate species. Receptors that respond to a
second form of GnRH (GnRH-II) have been
identified from mammalian and non-mammalian
species. It is known that Cys14 in the N-terminal
domain is connected to Cys199 in the second
extracellular loop, while Cys196 in this loop is
connected to the highly conserved Cys144 at the
extracellular end of transmembrane helix 3. There
are structural differences between non-mammalian
GnRH receptor and mammalian GnRHR type-I,
suggesting different ligand selectivity and
signal transduction pathways. In the present
study we mutated Cys199 in rat GnRH receptor to
Gly by site-directed mutagenesis. The mutation
was tentatively confirmed by restriction enzyme
mapping. It was found that the receptor with
mutation of Cys199 to Gly increased sensitivity
to mGnRH-I, but decreased sensitivity to
cGnRH-II, suggesting that Cys199 allows mGnRH to
bind to the receptor, while it impairs cGnRh-II
from binding to the receptor. (Supported by
NIH-FICMIRT Grant, TW00033-9, Barry University)
A Tentative Role of the Disulfide bridge between
Cysteine14 and Cysteine 199 in the GNRH Receptor
Activation and Ligand Selectivity
Empress Hughes1, Cheng Bing2, Jae Y. Seong2, Hyuk
B. Kwon2

Barry University, Florida, USA1 Hormone Research
Center, R. of Korea2
INTRODUCTION

• The CV-1 cells were seeded at 5x 1000 cells/well
  into 24-well plate one day before the
  transfection with the SuperFect (Qiagen, Genman).
  The transfection procedure is according to the
  one provided by the manufactor. In brief, 100ng
  of the receptor plasmid DNA and 200ng of the
  c-fos promoted plasmid DNA were diluted in the
  serum-free medium OPTIM (Lifetech), and from the
  transfection mixture with the SuperFect in room
  temperature for 5-10 minutes, and the mixture was
  transferred to the cell after washed with the
  warm DPBS. Incubate the cells with the mixture
  for 6 hours,and add another 500ul of the DMEM
  medium. Thirty-six hours after the transfection,
  the cells were starved in the serum-free DMEM for
  at 18 hours, and then the cells were treated with
  the GnRH in a series of the concentration
  indicate in the figure7 on the x-axis. The cells
  were harvested, washed with the ice-chilled PBS
  once, and lysed in 100ul of the lysis buffer for
  at 20 minutes. Thirty ul of the lysate was
  transferred into 96-well plate to measure the
  luciferase activity according the standard
  method. The luciferase activity was transformed
  into fold induction by
• fold induction absolute activity/ basal
  activity.
• Data were analysed using statistical analysis
  GraphPad PRISM(San Diego, CA,USA)

Gonadotropin-releasing hormone (GnRH) is a
decapeptide that is synthesized and secreted from
hypothalamic neurons (1,2). It is known to play
an important role in human reproduction and may
be used in cancer therapy. GnRH is an essential
reproductive stimulant in both males and females.
This decapeptide particularly stimulates
luteinizing hormone (LH) and follicle-stimulating
hormone (FSH), from the anterior pituitary, which
then stimulate gonadal secretion of testosterone,
progesterone, and estrogen, along with the
maturation of the oocytes in the ovaries,
testosterone, and sperm production in the testis.
GnRH receptors belong to the G protein coupled
receptor family (GPCR), which is characterized by
having seven transmembrane domains connected with
3 extracellular loops and 3 intracellular
loops,N-terminus and C-tail (Fig. 1) (3). Most
GPCR families have at least one conserved
cysteine which may form a disulfide bond.
Mutations of these cysteine residues disrupts
receptor activation and ligand binding. There
have been two disulfide bridges found to date
between Cys114 and Cys196 and between Cys14 and
Cys 199. Current literature states that the
disulfide bridge between Cys 114 and Cys 196 is
crucial for ligand binding and receptor
activations, while there have been limited
studies done on the role of the bridge between
Cys14 and Cys 199.
Fig. 5 In order to achieve this mutation we must
alter the DNA sequence from TGU that codes for
cysteine into TGU for glycine. This will alter
the restriction site for Pst1 and should provide
a different restriction enzyme pattern when
compared to wild type DNA.
1 2 3 4 5 6 M
Fig 6. M 100 bp DNA ladder. Lane 1 Plasmid DNA
from clone 1 Lane 2 Plasmid DNA from clone
2 Lane 3 Plasmid DNA from clone 3 Lane 4
Plasmid DNA from clone 4 Lane 5 Wild Type rat
GnRH receptor Lane 6 Plasmid DNA from clone
5 All samples were digested with Pst1
           

 
1kb
Disulfide Bridge
 

Fig. 1. Amino Acid Sequence of the human GnRH
receptor. Arrow indicate the disulfide bridge.
Cysteine in position 199 (in rat) was mutated to
Glycine.
1 kb ladder

OBJECTIVE
Fig2. The rat GnRH receptor was cloned into the
pcDNA (Invitrogen) at the restriction enzyme
sites EcoR I/Xba I, the primers PC-F/PC-R are the
sequence from the pcDNA. The primer RatC199-R are
the upstream sequence of the mutation site, and
the primer RatC199G-F is the DNA sequence with
the mutated nucleotide around the mutation site.
When PCRs are done using the plasmid DNA rat
GnRHR in pcDNA as a PCR template, the primers
work like the PC-F/RatC199-R to get the 5end
fragment without the mutation of the GnRH
receptor cDNA and PC-R/RatC199G-F to get the 3
end fragment with the mutation. To get the full
length of the cDNA with the mutation, the PCR is
done like that PCR product, using the above PCRs
as a template, and the primers are PC-F/PC-R.
The purpose of this study was to evaluate the
importance of the disulfide bridge between C199
and C14 in ligand binding. The objective was
achieved by mutating the Cysteine199 to
Glycine199 in the GnRH receptor.
MATERIALS AND METHODS
1 2 M
1 2 M
Fig 7. The effects of the rat GnRH receptor C199G
mutant on the ligand selectivity WT wild type
C199G GNRH receptor mutation mGNRH mouse
cGNRHchicken

• PCR conditions were denaturation at 94?C for 5
  min, followed by 25 cycles at 94?C for 40 sec.,
  at 53? C for 40 sec., and at 72? C for 1 min.
  PCR products of approximately 250 bp and 850 bp
  were expected.
• Primer Sequence
• PC-F/RatC199G-R 5 - CAC ACA TTG CGA GAA A
  - 3
• PC-R/RatC199G-F 5 - TTT TCG CAA TGT GTG
  ACC CAC GGC AGC - 3
• 1.2 Agarose gels were used. Plasmids were cut
  with appropriate enzymes, under standard
  conditions and electrophoresis was carried out at
  120volts for 15 minutes. They were examined under
  UV light and photographed using a digital camera.
  
• Plasmids were isolated using a QIAGEN MiniPrep
  kit.

1.5kb
1.5 kb
1.1 kb
500bp

• The GnRH receptor mutation from cysteine199 to
  glycine199 was achieved
• The GnRH receptor mutant shows decreased
  sensitivity for cGnRH-II
• The GnRH receptor mutant shows increased
  sensitivity for mGnRH-I
• Cysteine plays a role in allowing mGnRH-I to bind
  to the receptor
• Further research currently involves designing
  additional GnRH mutations as well as double
• mutations to further identify critical regions
  in receptor/ ligand binding.

Fig 3.M 100bp DNA ladder Lane 1 PC-F/
ratC199G-R Lane 2 PC-R/ rat C199G-R
Fig. 4 M 1 kb DNA ladder from Ellips. Lane 1
PCR product from the wild type plasmid. Lane 2
PCR product using primers PC-F/ PC-R.
REFERENCES
ACKNOWLEDGEMENTS

• 1) Sealfon, S.C., Weinstein H., and Millar R.P.
  (1997) Molecular Mechanisms of Ligand Interaction
  with the Gonadotropin-Releasing Hormone Receptor,
  Endocr. Review 1997 18(2) 180-205.
• 2) Kaiser U.B., Conn, P.M., and Chin W.W. (1997)
  Studies of Gonadotropin-Releasing Hormone (GnRH)
  Action Using GnRH Receptor-Expressing Pituitary
  Cell-Lines Endocr. Review 1997 18(1) 46-70.
• 3) Ott, T.,Troskie, B., Roeske, R., Illing, N.,
  Flanagan, C., Millar, R.(2002) Two Mutations in
  the Extracellular loop 2 of the Human GnRH
  Receptor Convert an Antagonist to an Agonist..
  Molecular Endocrinology 2002 16(5) 1079-1088.

Hormone Research Center, R. of Korea
Barry University, FL Dr. Gerhild Packert Dr.
Peter Lin Dr. Ana Jimenez Dr. Mary
Handel-Fernandez Dr. Flona Redway Sr. John Karen
Frei, O.P., Ph.D. NIH-FIC MIRT Grant
(TW00033-09), Barry University
Chen Bing Wang Seong Lab Dr. Jae Y. Seong Dr.
Hyuk B. Kwon