Functional characterization of two naturally occurring mutations V221G and T449N in the follicle stimulating hormone receptor

Accepted Manuscript 221 449 Functional characterization of two naturally occurring mutations V G and T N in the follicle stimulating hormone receptor Antara A. Banerjee, Swati K. Achrekar, Shaini Joseph, Bhakti R. Pathak, Smita D. Mahale PII:

S0303-7207(16)30489-0

DOI:

10.1016/j.mce.2016.11.020

Reference:

MCE 9734

To appear in:

Molecular and Cellular Endocrinology

Received Date: 15 September 2016 Revised Date:

21 November 2016

Accepted Date: 22 November 2016

Please cite this article as: Banerjee, A.A., Achrekar, S.K., Joseph, S., Pathak, B.R., Mahale, 221 449 S.D., Functional characterization of two naturally occurring mutations V G and T N in the follicle stimulating hormone receptor, Molecular and Cellular Endocrinology (2016), doi: 10.1016/ j.mce.2016.11.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Functional characterization of two naturally occurring mutations V221G and T449N in the

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follicle stimulating hormone receptor

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Antara A. Banerjeea, Swati K. Achrekarb, Shaini Josephb, Bhakti R. Pathaka, Smita D. Mahalea,b*

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a

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Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India

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b

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(Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India

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*Corresponding author at: Division of Structural Biology and ICMR Biomedical Informatics

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Centre, National Institute for Research in Reproductive Health (Indian Council of Medical

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Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India. Tel.: +91 22 24192001;

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Fax: +91 22 24139412.

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E-mail address: [email protected] (S.D. Mahale).

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Division of Structural Biology, National Institute for Research in Reproductive Health (Indian

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ICMR Biomedical Informatics Centre, National Institute for Research in Reproductive Health

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Keywords: Extracellular domain; FSH receptor; Naturally occurring mutations; Ovarian

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hyperstimulation syndrome; Primary amenorrhea

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Abstract :

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Naturally occurring mutations in follicle stimulating hormone receptor (FSHR) affect the

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receptor function. Here, we characterized two such previously reported mutations, V221G and

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T449N, in the extracellular domain and transmembrane helix 3, of FSHR, respectively. Functional

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studies with the V221G mutant demonstrated an impairment in FSH binding and signaling.

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Validation of X-ray crystallography data indicating the contribution of FSHR specific residues in

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the vicinity of V221 to contribute to FSH-FSHR interaction was carried out. In vitro mutational

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studies showed that these residues are determinants of both FSH binding and FSH induced

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signaling. Analysis of the T449N mutation revealed that it results in an increase in FSH binding

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and high cAMP response at lower doses of FSH. A marginal hCG induced and no TSH induced

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cAMP production was also observed. These findings corroborated with the clinical

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manifestations of primary amenorrhea (V221G) and spontaneous ovarian hyperstimulation

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syndrome (T449N) in women harbouring these mutations.

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Highlights

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39  Ligand binding and signaling ability tested in vitro for FSHR mutants V221G and T449N 40  Primary amenorrhea seems to be due to reduced FSHR activity in the V

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G mutation

41  FSHR specific residues around V221 interact with FSH and are crucial for signaling 42  sOHSS reported by our group in a pregnant woman is due to T449N mutation in FSHR gene 43

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Functional characterization of two naturally occurring mutations V221G and T449N in the

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follicle stimulating hormone receptor

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1.Introduction

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Follicle stimulating hormone (FSH), binds to and activates its cognate G-protein coupled

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receptor, the follicle stimulating hormone receptor (FSHR). FSHR is expressed on the granulosa

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cells of the ovary in females (Sanford and Batten, 1989) and Sertoli cells of the testes in males

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(Fletcher and Reichert, 1984). The signaling pathways that are triggered by the FSH-FSHR

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interaction are essential for folliculogenesis in females and maintenance of spermatogenesis in

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males (Dias et al., 2002). FSHR belongs to the subfamily of glycoprotein hormone receptors

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(GPHRs) along with the common receptor for luteinizing hormone/choriogonadotropin (LH/CG

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receptor) and the receptor for the non-gonadotropin thyroid-stimulating hormone (TSH receptor)

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(Salesse et al., 1991). These receptors are characterized by the presence of a large extracellular

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domain (ECD) made up of N-terminal leucine rich repeats followed by a hinge region which

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connects the ECD to the serpentine transmembrane domain (TMD). The leucine rich repeats

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mediate ligand binding with high affinity and specificity (Vassart et al., 2004). Following FSH

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binding to the leucine rich repeats of FSHR, a sulfotyrosine (sTyr) binding pocket is created in

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FSH into which the sulfated Tyr335 residue from the hinge region is inserted, resulting in FSHR

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activation (Jiang et al., 2012). The TMD of FSHR mediates signal transduction. The seven α-

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helices of the transmembrane domain are connected to each other by means of three extracellular

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and three intracellular loops. A short C-terminal tail is present in the cytoplasm.

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Several naturally occurring point mutations in FSHR manifesting into pathophysiological

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conditions have been reported (reviewed by Tao and Segaloff, 2009, Desai et al., 2013, Siegel et

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al., 2013). The site of the receptor mutation, that is, the domain in which the mutation is located,

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can be attributed to the function that gets affected, as a result, in its life cycle. For example,

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mutations in the ECD, I160T (Beau et al., 1998), A189V (Aittomäki et al., 1995), D224V (Touraine

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et al., 1999), P348R (Allen et al., 2003), result in impairment in hormone binding ability and

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subsequent diminished receptor function. One such heterozygous mutation, V221G, has been

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reported in a patient with primary amenorrhea (Nakamura et al., 2008). The residue affected

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here, V221, is also implicated in forming a hydrophobic pocket for FSH binding as reported by

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Jiang et al. in 2012. The FSH-FSHRED crystal structure reported by Jiang et al. (2012) indicates a

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wide-ranging hormone-receptor interface leading to a more compact hormone-receptor

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interaction. Other than the primary site of hormone binding (Fan and Hendrickson, 2005),

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several FSHR specific residues especially V221 (non conserved across the GPHRs) contribute to

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this. The presence of a naturally occurring mutation at this site along with its involvement in

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FSH binding as shown by crystallography studies signifies that Valine at 221 position is a crucial

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residue for FSH-FSHR interaction. Since the crystal structure is a partial one and provides only a

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molecular glimpse of the FSH-FSHR interacting sites, experimental validation of the residues

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predicted to be involved is necessary.

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On the other hand, the interhelical ionic locks between the helices of the transmembrane domain

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keep the receptor in a constrained, and thus, inactive state. Several reports have shown that

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mutation in the TMD not only can result in weakening of these ionic interactions (Montanelli et

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al., 2004b) but also promiscus activation of the receptor by the non-cognate ligands hCG and

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TSH (Vasseur et al., 2003, Montanelli et al., 2004b, De Leener et al., 2006). This can result in a

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life threatening complication known as ovarian hyperstimulation syndrome (OHSS) due to

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exogenous FSH administered in the setting of assisted reproduction program (iatrogenic OHSS).

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OHSS can also occur due to overproduction of hCG in the first trimester of pregnancy or high

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TSH levels as a result of hypothyroidism during pregnancy (spontaneous OHSS). One such

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novel heterozygous mutation T449N in the transmembrane helix 3 (TMH3) has been identified by

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our group in a patient exhibiting symptoms of sOHSS (Chauhan et al., 2015). Functional

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characterization of this naturally occurring mutation is necessary in order to establish genotype-

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phenotype association.

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Structure-function relationship studies by site directed mutagenesis approach is useful in

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characterizing the functional consequences of mutations in FSHR. In the present study, the effect

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of the naturally occurring mutations V221G and T449N on FSH receptor function was determined.

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We also identified the FSHR residues in close proximity of V221 shown to be involved in

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interaction with FSH from the FSH-FSHR complex structure. Hence, we proposed to check for

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the contribution of the residues K146, E197, I222 and K243 in conferring functional specificity to

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FSHR. This was done by swapping with the corresponding residues from the closely related

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LH/CGR as these closely related GPHRs can coexist in mature ovarian granulosa cells. The

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effect of the naturally occurring and induced mutations on FSH receptor expression, FSH

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binding ability and the signaling response generated thereafter was investigated. This provided

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insights into the contribution of the above mentioned residues in FSH receptor function.

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2. Materials and methods

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2.1 Residues of FSHR selected for site directed mutagenesis

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The naturally occurring and induced mutations chosen for in vitro functional studies are listed in

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Table 1.

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Naturally occurring mutations

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Mutant FSHR V221G was selected to identify the cause of impairment in the mutant receptor in

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the patient with primary amenorrhea. The woman with primary amenorrhea harbouring the

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V221G mutation in FSHR (Nakamura et al., 2008) had moderately developed secondary sexual

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characteristics, normal sized ovaries but small antral follicles. Her serum FSH levels were not

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high and serum estradiol levels were low but detectable. Only administration of high doses of

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human menopausal gonadotropin (hMG) triggered follicular growth. This indicated that the

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follicles had the potential to respond to high doses of gonadotropin and develop to the antral

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stage. The ovarian response to high dose hMG stimulation suggested an FSHR dysfunction of

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her ovaries.

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The T449N substitution was selected for delineation of the effect of the mutation in the receptor in

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the patient presenting symptoms of sOHSS during her first pregnancy (Chauhan et al., 2015).

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She had a normal thyroid profile and normal levels of hCG β but elevated estradiol levels of

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12927 pg/dl (normal range 188-2497 pg/dl). Multiloculated cystic lesion was revealed by

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ultrasonography. Ascites and bilateral pleural effusion were also observed. Magnetic resonance

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imaging confirmed the features of OHSS.

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Induced mutations

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The 3-D model of FSH-FSHRED complex as reported by Jiang et al., 2012 (PDB ID: 4AY9) was

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visualised using Discovery studio 3.5 (Accelrys, Inc., San Diego, CA, USA). Residues in close

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proximity to V221 of FSHR which interacted with FSH were selected for further studies. The

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ECD mutants K146N, E197S, V221G, V221K, I222T and K243R were subjected to Protein interactions

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calculator (PIC) server analysis (Tina et al., 2007). Mainly the hydrophobic, ionic, main chain-

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main chain and main chain-side chain interactions that differed between the WT receptor and the

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mutants were studied. All the mutants including T449N were also subjected to SIFT (Sorting

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Intolerant from Tolerant) analysis which predicts the propensity of amino acid substitutions to

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affect protein structure, and hence function, prior to carrying out functional validation (Sim et al.,

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2012).

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2.2 Construction of FSHR mutants

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The substitution mutants were generated by site-directed mutagenesis as mentioned in

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Dupakuntla et al. (2012). hFSHR cloned into pSG5 vector was used as the template for

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mutagenesis (Dias et al., 2010). DNA sequencing was carried out to confirm the mutations. The

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plasmid DNA extraction of mutant constructs for transfection was then carried out using a midi-

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prep kit (Sigma, St. Louis, MO, USA).

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Chinese hamster ovary (CHO) cells were used as the transfection host. CHO cells were obtained

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from the Cell Repository at the National Centre for Cell Science, Pune, India, and were

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maintained in DMEM-F12 (Gibco; Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal

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bovine serum (Gibco; Invitrogen, Carlsbad, CA, USA) and antibiotics. Transient transfection of

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wild type FSHR and the substitution mutants was performed using Lipofectamine and Plus

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reagents (Invitrogen, San Diego, CA, USA) as per manufacturer’s protocol. All the assays were

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performed 48 h post transfection. Cells transiently transfected with empty vector pcDNA 3.1+

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which did not encode FSHR served as the negative control.

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2.4 Determination of FSHR expression by western blotting

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Western blotting was performed to determine total FSHR expression in transfected cells.

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1.5X105 of CHO cells were seeded per well in a 24 well plate. Twenty four hours post seeding,

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cells were transfected with 500 ng of WT or mutant FSHR plasmid constructs diluted in Opti-

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MEM (Invitrogen, San Diego, CA, USA) using Lipofectamine and Plus reagents. The two

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naturally occurring mutations, V221G and T449N were heterogyzous in nature. Hence, assuming

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what is possibly the patient’s physiological milieu of FSH receptors, cells were co-transfected

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with equal amounts of WT and each of the mutant constructs (WT/V221G and WT/T449N).

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Forty eight hours post transfection, cell lysates were prepared using lysis buffer containing 1%

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Igepal, 0.4% sodium deoxycholate, 10 mM Tris pH 7.5, 6.6 mM EDTA, 140 mM sodium

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chloride and protease inhibitors. Protein lysates (20 µg) were then loaded on 7.5% SDS-PAGE

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followed by electrophoretic transfer on PVDF membrane (Immobilon-P, Millipore, Bedford,

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MA, USA) using a semi-dry transfer apparatus (Bio-Rad, Richmond, CA, USA). FSHR was

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detected using a monoclonal antibody MAb 106.105 (1:1000 dilution; l µg/µl) which was a kind

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gift from Dr. J.A. Dias, Wadsworth Centre, NY, USA. The blot was also probed with MAb

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against β-actin (Santa Cruz Biotechnology, CA, USA) which served as a loading control. The

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secondary antibody used was goat anti-mouse IgG conjugated to horseradish peroxidase (1:2000

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dilution; Santa Cruz Biotech, Santa Cruz, CA, USA). Detection of protein bands was performed

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by chemiluminescence method using enhanced chemiluminescence (ECL) Western blotting

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detection reagents (GE Healthcare, Buckinghamshire, UK). The membranes were then exposed

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to X-ray film (Kodak, NY, USA).

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2.5 Confirmation of co-transfection of WT/mutant receptor constructs for heterozygous

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patient mutations

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In order to confirm the expression of both the WT and mutant receptor constructs in the co-

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transfection experiments, RNA was extracted from cells (3X105 of CHO cells in 35-mm dishes)

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transfected with 2μg of WT, V221G, T449N, empty vector pcDNA 3.1 + (negative control) and co-

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transfected with 1μg each of WT and V221G or WT and T449N constructs using TRIZOL

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(Invitrogen, Carlsbad, CA). cDNA was synthesized using Superscript III reverse transcriptase

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enzyme and oligo deoxythymidine (dT) as primers (Invitrogen) as per the manufacturer’s

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instructions. PCR amplification was then carried out using FSHR primers and the products

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obtained were subjected to automated DNA sequencing.

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2.6 Cell surface FSHR expression by direct immunofluorescence and flow cytometry

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Cell surface FSHR expression was determined by direct immunofluorescence on transiently

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transfected CHO cells seeded on cover slips in a 6-well plate. The protocol used was modified

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from Meduri et al. (2003). The FSHR antibody, MAb 106.105, was labeled with Alexa Fluor 488

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using a MAb labeling kit (Molecular Probes, Life Technologies, Carlsbad, CA, USA). Cells were

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fixed using 4% paraformaldehyde for 10 minutes at RT, blocked with 5% BSA containing PBS

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for 30 mins at RT followed by incubation with Alexa Fluor 488 labeled-MAb 106.105 for 2 h at

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RT in dark. Thereafter, cells were rinsed with PBS and nucleus was stained using DAPI for 10

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minutes at RT in dark. Cells were then mounted using Vectashield (Vector Laboratories,

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Burlingame, CA, USA). Images were collected using an oil immersion 63X objective with NA

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1.4 on a LSM510-Meta confocal system (Carl Zeiss, Jena, Germany).

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Flow cytometry was performed to quantitate cell-surface FSHR expression using a FACS Scan

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Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) as described previously

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(Dupakuntla et al., 2012).

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2.7 Binding of

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I-FSH by radioreceptor assay

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Binding of

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was assessed by carrying out radioreceptor assay in 24 well plates (Dias et al., 2010). The

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radioiodination of hFSH (procured from National Hormone and Pituitary Programme, NIDDK,

I-FSH to the transiently transfected WT or point mutants of FSHR in CHO cells

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Bethesda, MD, USA) was performed by the iodogen method (Fraker and Speck, 1978). Forty

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eight hours post transfection, growth medium was replaced with 250 µl of fresh serum free

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medium to which 50 µl of serum free medium containing 200 ng of

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specific activity 16-20 µCi/µg) was added and incubated at RT for 1 h in the absence or presence

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of unlabeled FSH (1 µg/well) to determine non-specific binding. Displacement assays were also

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performed where the transfected cells were incubated in the presence of increasing

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concentrations of unlabeled FSH (0.1-1000 ng). After 1 hour, cells were washed twice with

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chilled PBS on ice. Surface bound labeled hormone was then eluted by incubating cells with 300

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µl of elution buffer (50 mM glycine/100 mM NaCl, pH 3.0) for 10 min on ice and collecting the

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supernatant in tubes. Internalized hormone was measured by washing cells with 1 ml of PBS and

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then solubilizing in 200 µl of 2 N NaOH for 1 h at room temperature. The surface bound and

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internalized counts were measured on an automatic γ-counter (Wallac 1470, WIZARD, Turku,

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Finland).

I-FSH (1X106 cpm,

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Displacement assays were also performed in the presence of unlabeled LH or unlabeled hCG to

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determine receptor specificity.

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2.8 cAMP production

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cAMP production in CHO cells transiently transfected with WT and point mutants of FSHR was

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estimated using a commercially available enzyme immunoassay (EIA) kit (Cayman Chemical

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Company, Ann Arbor, MI, USA). Forty eight hours post transfection, cells were incubated with

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serum free DMEM medium containing 0.1 mM of 3-isobutyl-1-methylxanthine (IBMX), at 37°C

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for 15 mins followed by stimulation with increasing doses of hFSH (1 ng, 10 ng and 100 ng) for

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1 h. The highest dose of 100 ng FSH was selected based on preliminary experiments performed

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with WT FSHR where the cAMP generated was maximum.Basal cAMP (0 ng FSH) values were

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also determined. After 1 h, cells were lysed with 0.1 M HCl and centrifuged at 1000 g for 10

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mins. Supernatants were diluted in assay buffer provided with the kit and subjected to EIA

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according to the instructions provided by the manufacturer. cAMP production was also estimated

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in response to stimulation by hCG (10 ng, 100 ng, 500 ng and 1000 ng), TSH (10 ng and 100 ng)

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and LH (10 ng and 100 ng) for 1 h at 37°C.

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2.9 Statistical analysis

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Statistical analysis was performed using the unpaired Student’s t-test from the Software

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‘GraphPad Prism 5.0’ (GraphPad Software, Inc., San Diego, CA, USA). The graphs either

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represent data expressed as mean ± S.E.M. of three independent experiments performed in

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duplicates or mean ± S.E.M. of a single representative experiment performed in duplicates as

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indicated in the figure legends. The values of *P<0.05, **P<0.01, and ***P<0.001 with respect

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to WT FSHR were considered to be statistically significant.

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3. Results

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3.1 Visualisation of 3-D representation of FSH-FSHRED complex and in silico analysis of

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FSH-FSHR interactions in mutant receptors

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The 3-D representation of FSH-FSHRED complex is shown in Fig 1. Apart from the residue V221,

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certain FSHR specific residues in its close vicinity implicated to be contributing to the interface

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of FSH-FSHRED were chosen for mutagenesis studies and their positions in the ECD are

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indicated. Table 1 lists the mutations generated along with the basis for its selection for

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mutagenesis studies.

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The type of interactions observed in the wild type receptor as seen by Protein Interactions

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Calculator (PIC) analysis are tabulated in Table 2. Importantly, the FSH-FSHR interaction

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interface involving hydrophobic interactions around Val at 221 position were seen to be lost in

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both the V221G and V221K mutants. Similar disruption of hydrophobic interaction for the I222T

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mutant and ionic interaction for the E197S mutant was observed as compared to WT FSHR.

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Also, modifications in the structure of FSHR, which in turn affect the function, were gauged

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using the prediction tool SIFT. Prediction for the T449N mutation was found to be ‘damaging’

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whereas for all other mutations it was found to be ‘tolerant’.

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3.2 Creation of substitution point mutants of FSHR and confirmation of co-transfection of

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WT/mutant receptors for the heterozygous patient mutations

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The mutant FSHR constructs K146N, E197S, V221G, V221K, I222T, K243R and T449N were

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generated. Full length DNA sequencing was carried out to confirm the nucleotide substitution

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resulting in desired mutation and to ensure the absence of other undesired mutations.

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For the heterozygous patient mutations, DNA sequencing following PCR amplification using

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FSHR primers from cells co-transfected with WT/V221G and WT/T449N mutant constructs

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revealed two overlapping peaks indicating the presence of both the WT and mutant FSH

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receptors (data not shown).

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3.3 Determination of total FSHR expression

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Total FSHR expression was determined in the lysates from CHO cells transiently transfected

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with the WT and mutant FSHR constructs by Western blotting. All the mutant receptors showed

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comparable FSHR expression to WT FSHR (Figures 2A and 2B). Equal beta-actin expression in

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all lanes ensured equal protein loading.

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3.4 Determination of cell surface FSHR expression

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In order to be functional, the FSH receptor should be able to traffick to the cell surface.

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Therefore, determination of the cell surface FSHR expression of the mutant receptors is a

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prerequisite to the measurement of the hormone binding ability.

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To determine the cell surface FSHR expression of WT and its mutants, confocal microscopy was

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performed using FSHR MAb 106.105 conjugated to Alexa 488 as a probe by direct

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immunofluorescence method. Quantitative analysis was also carried out for the same by flow

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cytometry.

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immunofluorescence studies revealed similar FSHR expression in WT and all the FSHR mutants

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generated (Figure 3). No FSHR expression was detected in cells transfected with the empty

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vector pcDNA 3.1+.

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Qualitative

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A quantitative proof of the same was obtained by flow cytometry experiments (Figures 4A &

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4B). The mean fluorescence intensity of all the mutant receptors compared with that of the WT

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FSHR (considered to be 1) revealed no significant differences. Hence, these mutations did not

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alter the FSHR expression. The relative values of the cell surface FSHR expression of the mutant

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FSH receptors with respect to WT FSHR (considered to be 100 %) have been listed in Table 3.

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3.5 Radioreceptor assay to determine binding of FSH to FSHR

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In order to evaluate the effect of the FSHR mutations on FSH binding, radioreceptor assay was

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performed. Quantitation of the fraction of cell surface bound and internalized hormone receptor

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complexes was carried out. For this, the cells expressing the WT FSHR or the mutant receptors

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were incubated with

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unlabeled FSH under non-equilibrium conditions. Though the substitutions at 221 and 449

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positions did not alter the cell surface FSHR expression, the effect on hormone binding ability

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varied (Figure 5A). The V221G mutation showed a significant reduction (two-fold decrease) in

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both the surface bound and internalized amounts of FSH-FSHR complex as compared to WT

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FSHR. On the other hand, T449N FSHR showed an enhancement (upto two-fold) in surface

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bound and internalized hormone-receptor complexes. For the co-transfections of WT/V221G and

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WT/ T449N, the phenotype observed was intermediate between that observed for the WT receptor

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and the respective homozygous mutations. FSH binding and FSH-FSHR internalization of the

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K146N mutant was found to be similar to that observed for WT FSHR, for the mutations E197S,

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V221K and I222T it was significantly low whereas it was significantly higher for K243R mutation

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as compared to WT. The amount of internalized ligand was expressed as a percentage of the total

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specific binding for each of the receptors (Figure 5B). No significant differences were observed.

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A dose-response equilibrium binding assay was also carried out with a fixed amount of 125I-FSH

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and increasing doses of unlabeled FSH. No significant differences were observed in the FSH

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binding affinities in the V221G, T449N or the other mutant FSH receptors as compared to WT

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receptor (Figure 6A and 6B). The

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have been tabulated (Table 3).

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I-FSH binding parameters of the WT and mutant receptors

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No displacement of 125I-FSH could be detected in a radioreceptor assay in which cells expressing

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the WT FSHR or the point mutants were incubated with

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concentrations of unlabeled LH or hCG. This indicated that the ligand binding specificity was

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retained by the mutant receptors inspite of the amino acid substitutions.

I-FSH in the presence of saturating

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3.6 FSH induced cAMP production

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The cAMP production of WT FSHR and its substitution point mutants was evaluated without

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(basal levels) or with stimulation with 1 ng. 10 ng and 100 ng FSH for one hour (Figure 7). The

358

basal cAMP levels were similar and low in all mutants similar to WT FSHR. Consistent with the

359

FSH binding studies, the cAMP response of the V221G and WT/V221G was found to be

360

significantly lower than WT FSHR at both the doses of FSH. Interestingly, the T449N and

361

WT/T449N mutations showed a significantly higher cAMP response at the low dose of FSH (10

362

ng). However, at higher doses of 100 ng FSH, the response was similar to that observed for WT

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FSHR. Similar results were obtained with the K243R mutant. For the E197S, V221K and I222T

364

mutants the cAMP response was low, whereas that for K146N mutation was similar to that of wild

365

type receptor. None of the mutants including, T449N and WT/T449N showed a high basal response

366

as compared to WT FSHR indicating that these mutations are not constitutively active in nature.

367

The maximal cAMP response at 100 ng dose of FSH (Emax at 100 ng of FSH) and EC50 values for

368

the WT and mutant FSH receptors are listed (Table 3).

3.7 Determination of hCG/TSH/LH induced cAMP response

M AN U

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369

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363

371

cAMP production (if any) of the mutant receptors was also evaluated in response to stimulation

373

with the other glycoprotein hormones hCG, TSH, and LH. In sOHSS, the mutant receptor loses

374

specificity and responds to high levels of pregnancy induced hCG. At the doses of 10 ng and 100

375

ng, no hCG-induced cAMP response was observed in the mutant receptors. However at the

376

higher doses of 500 ng or 1000 ng, a subtle, yet statistically significant cAMP response was

377

observed for the T449N and WT/T449N mutations as compared to WT FSHR (Figure 8A). The

378

T449N mutation showed a high cAMP response even at a low dose of FSH as compared to WT

379

FSHR. However, very high doses of hCG were required for the indiscriminate activation of this

380

mutant receptor and the cAMP production was minimal.

381

To detect if the mutations in FSHR had resulted in their responsiveness to TSH, cAMP response

382

of the WT FSHR and the mutants was estimated post stimulation with TSH. However, no TSH

383

induced cAMP response was observed in cells expressing the WT FSHR, T449N mutant or any of

384

the other mutant receptors (Figure 8B).

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In case of the LH/CGR swap, whether the substitutions resulted in the mutant receptor gaining

387

responsiveness to LH had to be discerned. No LH induced cAMP response (Fig. 8C) was

388

observed in cells expressing either mutant or WT FSHR. Therefore the substitutions in FSHR

389

did not alter the specificity of the receptor towards LH.

390

4. Discussion

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392

The relevance of the study of FSHR residues is strengthened by the fact that many naturally

394

occurring mutations have been reported in the different domains of the receptor (Aittomaki

395

et al.,1995, Beau et al., 1998, Touraine et al., 1999, Doherty et al., 2002, Meduri et al., 2003,

396

Vassuer et al., 2003, Montanelli et al., 2004a, Tranchant et al., 2011, Casas-González et al.,

397

2012, Uchida et al., 2013, Desai et al., 2015, Bramble et al., 2016). These exhibit varied

398

phenotypes like ovarian dysgenesis, primary amenorrhea or primary ovarian failure in case of

399

inactivating mutations. On the other hand, several activating mutations resulting in ovarian

400

hyperstimulation syndrome also have been reported which can either be iatrogenic (Desai et al.,

401

2015) or spontaneous in nature (Vassuer et al., 2003, De-Leener et al., 2006). Two of such

402

naturally occurring mutations, V221G and T449N, reported in a woman with primary amenorrhea

403

and in a woman with spontaneous ovarian hyperstimulation syndrome, respectively, have not yet

404

been characterized. Functional significance of these residues was investigated in the present

405

study.

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406 407

The V221G mutation in FSHR was reported by Nakamura et al. in 2008 in a patient with primary

408

amenorrhea.The underlying dysfunction in the receptor which resulted in this clinical condition 18

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was ascertained in vitro. Cell surface FSHR expression was unaltered by the substitution of

410

glycine at 221 position as evidenced by immunofluoresence and flow cytometry studies. This

411

suggested that the substitution did not affect any post translational modifications like

412

glycosylation (Davis et al., 1995) which are essential for proper folding and cell surface

413

trafficking of the mature receptor. However, FSH binding was impaired with a concomitant

414

impairment in internalization of FSH-FSHR complex in the V221G mutant receptor as compared

415

to WT FSHR. Subsequently, FSH induced cAMP response was also found to be significantly

416

low in cells transiently expressing the V221G mutant receptor. Therefore the diminished receptor

417

function due to this mutation can be attributed to the clinical manifestation of primary

418

amenorrhea. In case of the co-transfection experiments of WT and V221G FSHR to mimic the

419

patient’s repertoire of FSH receptors, the effect of the mutation on impairment in receptor

420

function was less pronounced due to the pool of WT FSHR which could rescue the function to

421

some extent.

SC

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422

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409

As V221 is present at the interface of FSH-FSHRED complex, we explored the contribution of

424

residues in its close proximity in FSHR function. The crystal structure of FSH-FSHRED complex

425

(Jiang et al., 2012) has identified a greater interface of FSH-FSHR interaction involving several

426

non conserved FSHR residues in the ECD, namely E197, V221, I222, and K243 which line this

427

interface (Jiang et al., 2014). Especially the Val at 221 position which interacts with P42 and A43

428

of L2β loop of FSH and Ile at 222 position are implicated in forming a hydrophobic pocket for

429

FSH binding and forming a second FSH-FSHR interaction site. The presence of the naturally

430

occurring mutation V221G at this position, which disrupts receptor function, emphasizes its

431

significance. In silico PIC analysis also revealed that the hydrophobic interactions involving Val

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at 221 and Ile at 222 with the surrounding residues of L2β loop of FSH are lost due to the

433

substitution. Also, Núñez Miguel et al. (2008) have speculated a clear role for the FSH receptor

434

specific residue K243 in conferring functional specificity to the receptor; whereas the role of the

435

K146, V221 and I222 residues in doing so is unclear. The importance of these residues is

436

underscored by the fact that they interact with residues in L2β loop of FSH. The residues in L2β

437

loop of FSH have been shown earlier to confer conformational stability to FSH α- and β-

438

subunits and in turn facilitate interaction of the heterodimeric FSH with FSHR (Roth and Dias,

439

1995 and 1996). Previously, our group has demonstrated that the non conserved or receptor

440

specific residues in the extracellular loop 2 of FSHR are indispensable for FSH mediated

441

internalization and activation to bring about signal transduction (Dupakuntla et al., 2012,

442

Banerjee et al., 2015). This underpins the importance of studying the receptor specific residues in

443

conferring functional specificity. Studies on chimeric FSHR/TSHR constructs (Schaarschmidt et

444

al., 2014) have emphasized the importance of the residue K243 in FSHR in conferring ligand

445

binding specificity. Mutation in the corresponding residue E251 in TSHR results in an attenuation

446

of the signaling response (Chen at al., 2010). These facts provided compelling evidence to test

447

the hypothesis that the residues K146, E197, V221, I222, and K243 act as determinants of specificity

448

of FSH receptor function. Towards this, we carried out mutagenesis studies by substitution with

449

the corresponding residues from the closely related LH/CGR. Thus, K146N, E197S, V221K, I222T,

450

and K243R mutants were generated, and further characterized.

SC

M AN U

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EP

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451

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432

452

Cell surface FSHR expression in the LH/CGR swap mutants K146N, E197S, V221K, I222T and

453

K243R revealed no significant differences. No significant differences in FSH binding affinities

454

among the mutant receptors was observed. But estimation of the amount of surface bound and

20

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internalized FSH-FSHR complex revealed that the E197S, V221K, I222T mutants exhibited

456

impairment in binding as well as internalization of FSH-FSHR complex. cAMP production was

457

also found to be low. The mutant K243R on the other hand, showed an increase in binding and

458

internalization of FSH-FSHR complexes and high cAMP production at lower doses of FSH

459

compared to WT FSHR. This indicated that the residues E197, V221, I222 and K243 indeed

460

contribute to the FSH-FSHR interaction. Further, in addition to the confirmation of crystal

461

structure data predicting their contribution in ligand binding, this study also shows that these

462

residues are crucial for signaling. K146N served as a negative control for the mutagenesis studies

463

as it behaved similar to WT FSHR in terms of FSH binding and FSH induced cAMP production.

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455

464

The second naturally occurring mutation, T449N, in TMH3 of FSHR, resulted in the patient

466

presenting symptoms of spontaneous OHSS during her first pregnancy (Chauhan et al., 2015).

467

Functional validation of this mutation disclosed interesting results. Inspite of comparable cell

468

surface FSHR expression as WT FSHR, significantly high binding of FSH and internalization of

469

FSH-FSHR complexes was observed. In accordance with the results of radioligand binding

470

studies, the T449N mutant showed a remarkable increase in cAMP production as compared to WT

471

FSHR. The increase in cAMP response post stimulation with 10 ng FSH was augmented.

472

However, at the higher dose of 100 ng, it was found to be similar to that observed for WT FSHR.

473

It is important to note that this mutant did not display high levels of basal cAMP production

474

ruling out the possibility that it is a constitutively activating mutation. Since the patient with the

475

T449N mutation had normal levels of hCG beta, FSH and TSH, the underlying trigger for

476

exhibiting sOHSS can be attributed to the mutation in FSHR. Thus, as per the classification

477

system for sOHSS laid down by De Leener et al. (2006), this is a case of Type I sOHSS. This

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type is characterized by the patient exhibiting symptoms of sOHSS in the first trimester of

479

pregnancy inspite of normal levels of hCG beta as seen in the patient here (Chauhan et al., 2015)

480

and also in the I545T mutation in TMH5 of FSHR reported by De Leener et al. (2006). Similar to

481

the results of co-transfection experiments with V221G mutant, the enhancement in FSHR function

482

in the heterozygous condition WT/T449N emulating the patient’s physiological pool of FSH

483

receptors was less conspicuous.

RI PT

478

SC

484

The cell surface expression of the WT/mutant receptors determined using a monoclonal antibody

486

MAb 106-105 directed against an epitope in the hinge region of FSHR (residues 300-315,

487

Lindau-Shepard et al., 2000) did not reveal any significant differences between the WT and

488

mutant FSH receptors. However, binding of

489

differences not in the affinity but in the capability of the amount of 125I-FSH maximally bound. It

490

appears that the % of cell surface receptor capable of binding to the ligand are altered although

491

the total surface expression of FSHR detected by antibody is similar in WT/mutants. Similar

492

results have been reported for FSHR mutants showing similar FSH binding affinity but

493

differences in the amount of surface bound or internalized hormone-receptor complexes eg: 5A-

494

FSHR (Kara et al., 2006), C-tail Cys mutants of FSHR (Uribe et al., 2008), D550A (Kluetzman et

495

al., 2011). In case of the naturally occurring mutants R573C (Beau et al., 1998), L601V (Touraine

496

et al., 1999), A419T (Doherty et al., 2002), inspite of comparable FSHR expression and FSH

497

binding affinity, altered FSH-induced cAMP levels were observed as reported by us. A plausible

498

explanation for this discrepancy could be that conformational changes in the receptor owing to a

499

mutation can have distinct functional effects. Similar FSH binding affinity but low or high

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125

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I-FSH to WT/mutant FSH receptors revealed

22

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500

cAMP generation ability could be due to the altered capability of mutant receptors to release the

501

inhibitory constraint required for its activation (Smits et al., 2003).

502

In order to determine if the FSHR mutations had resulted in their indiscriminate responsiveness

504

to the non-cognate ligands, hCG, TSH and LH, cAMP production was also measured in response

505

to these ligands. Only the T449N and WT/ T449N mutants exhibited a marginal cAMP response to

506

very high doses of hCG. Similar results for hCG and TSH induced activation was obtained for

507

the TMD mutations T449A/I (Montanelli et al., 2004b) and I545T (De Leener et al., 2006). Thus,

508

the threonine residue at 449 position indeed seems to be crucial for constraining the receptor to

509

an inactive state and preventing promiscus activation of FSHR by the non cognate ligands hCG

510

and TSH. This is reinforced by the presence of three naturally occurring mutations (T449A/I ,

511

Montanelli et al., 2004b and T449N, Chauhan et al., 2015) reported so far which result in

512

relaxation of this specificity barrier and development of ovarian hyperstimulation syndrome in

513

patients harbouring these mutations. Also, sOHSS can result from acquisition of sensitivity to

514

high TSH levels due to the hypothyroidism associated with a natural conception (Borna and

515

Nasery, 2007). This was however not the case with the patient with the T449N mutation who had

516

a normal thyroid profile (Chauhan et al., 2015). Consistent with this fact, no TSH induced cAMP

517

response was observed in cells expressing the T449N mutant receptor. Therefore, the decrease in

518

specificity of the T449N mutant was only restricted to hCG.

SC

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519

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503

520

In conclusion, two naturally occurring mutations V221G and T449N in the follicle stimulating

521

hormone receptor were studied. The diminished receptor function in terms of FSH binding and

522

cAMP signaling seems to be responsible for primary amenorrhea in the woman harbouring the

23

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V221G mutation. Selected FSHR specific residues in the vicinity of V221 were also swapped with

524

the corresponding residues from luteinizing hormone/choriogonadotropin receptor. In silico

525

analysis followed by in vitro studies indicated that these residues contribute to FSH-FSHR

526

interaction interface. On the other hand, an enhancement in the FSHR function in terms of

527

hormone binding and hormone induced signaling response in case of the T449N mutation

528

corroborates with the clinical manifestations of spontaneous ovarian hyperstimulation syndrome.

529

For the mutants V221G and T449N which are heterozygous in nature, cotransfection of equal

530

amounts of WT/ V221G and WT/ T449N displayed a phenotype intermediate between the WT

531

FSHR and V221G or T449N respectively. This provides further evidence for oligomerization of

532

FSH receptor (Thomas et al., 2007) and supports the notion that functional rescue of mutant

533

receptors takes place by di/oligomerization with WT FSHR as shown by Zarinan et al., 2010.

534

Thus, investigation of the follicle stimulating hormone receptor resulted in better understanding

535

of the pathophysiology associated with the loss of function mutation V221G and the gain of

536

function mutation T449N.

537

539

Acknowledgments

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538

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523

The research work in this study was supported by Grants received from the Indian Council of

541

Medical Research (NIRRH/RA/407/08-2016 and BIC/12(10)/2013), Government of India. The

542

help provided by Dr. S. Mukherjee, in-charge of DNA sequencing and Flow Cytometry facility

543

and the technical expertise of Ms. N. Joshi for DNA sequencing and Ms G Shinde and Ms S

544

Khavale for the assistance provided with flow cytometry experiments is acknowledged. We also

545

thank Dr. N. Balasinor, in-charge of Confocal Facility and Ms. S. Sonawane and Ms. R. Gaonkar

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for all the assistance provided. We acknowledge the kind gift of recombinant hTSH from Dr. M

547

G R Rajan and Ms. Bharati Jain, Radiation Medicine Centre (RMC) of Bhabha Atomic Research

548

Centre (BARC), Mumbai. The award of Senior Research Scholarship to Ms. Antara Banerjee by

549

the Lady Tata Memorial Trust, India, is gratefully acknowledged.

550 551

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657

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spontaneous ovarian hyperstimulation syndrome. J. Clin. Endocrinol. Metab. 89, 1255-1258.

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Montanelli, L., Van Durme, J.J., Smits, G., Bonomi, M., Rodien, P., Devor, E.J., Moffat-Wilson,

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K., Pardo, L., Vassart, G., Costagliola, S., 2004b. Modulation of ligand selectivity associated

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with activation of the transmembrane region of the human follitropin receptor. Mol. Endocrinol.

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18, 2061-2073. doi: 10.1210/me.2004-0036.

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Nakamura, Y., Maekawa, R., Yamagata, Y., Tamura, I., Sugino, N., 2008. A novel mutation in

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exon8 of the follicle-stimulating hormone receptor in a woman with primary amenorrhea.

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Gynecol. Endocrinol. 24,708-712. doi: 10.1080/09513590802454927.

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Núñez Miguel, R., Sanders, J., Chirgadze, D.Y., Blundell, T.L., Furmaniak, J., Rees Smith, B.,

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2008. FSH and TSH binding to their respective receptors: similarities, differences and

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implication for glycoprotein hormone specificity. J. Mol. Endocrinol. 41,145-164. doi:

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10.1677/JME-08-0040.

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Roth, K.E., Dias, J.A., 1995. Scanning-alanine mutagenesis of long loop residues 33-53 in

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follicle stimulating hormone beta subunit. Mol. Cell. Endocrinol. 109, 143-149. doi:

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Roth, K.E., Dias, J.A., 1996. Follitropin conformational stability mediated by loop 2 beta effects

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follitropin-receptor interaction. Biochemistry 35, 7928-7935. doi: 10.1021/bi952566j.

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Salesse, R., Remy, J.J., Levin, J.M., Jallal, B., Garnier, J., 1991. Towards understanding the

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glycoprotein hormone receptors. Biochimie. 73, 109-120. doi:10.1016/0300-9084(91)90083-D.

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Sanford, J. C., Batten, B. E., 1989. Endocytosis of follicle-stimulating hormone by ovarian

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granulosa cells: analysis of hormone processing and receptor dynamics. J. Cell. Physiol. 138,

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region and its adjacent domains on binding and signaling patterns of the thyrotropin and

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follitropin receptor. PLoS. One. 9, e111570. doi: 10.1371/journal.pone.0111570.

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Siegel, E.T., Kim, H.G., Nishimoto, H.K., Layman, L.C., 2013. The molecular basis of impaired

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follicle-stimulating hormone action: evidence from human mutations and mouse models. Reprod.

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Sim, N.L., Kumar, P., Hu, J., Henikoff, S., Schneider, G., Ng, PC., 2012. SIFT web server:

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predicting effects of amino acid substitutions on proteins. Nucleic Acids Res. 40, W452-W457.

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doi: 10.1093/nar/gks539.

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Tao, Y.X., Segaloff, D.L., 2009. Follicle stimulating hormone receptor mutations and

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reproductive disorders. Prog. Mol. Biol. Transl. Sci. 89,115-131. doi: 10.1016/S1877-

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Thomas, R.M., Nechamen, C.A., Mazurkiewicz, J.E., Muda, M., Palmer, S., Dias, J.A., 2007.

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proteolytic processing. Endocrinology 148, 1987-1995. doi: 10.1210/en.2006-1672.

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Tina, K.G., Bhadra, R., Srinivasan, N., 2007. PIC: Protein Interactions Calculator. Nucleic Acids

721

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Touraine, P., Beau, I., Gougeon, A., Meduri, G., Desroches, A., Pichard, C., 1999. New natural

724

inactivating mutations of the follicle-stimulating hormone receptor: correlations between

725

receptor function and phenotype. Mol. Endocrinol. 13, 1844-1854. doi:10.1210/me.13.11.1844.

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Tranchant, T., Durand, G., Gauthier, C., Crépieux, P., Ulloa-Aguirre, A., Royère, D., Reiter, E.,

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2011. Preferential β-arrestin signalling at low receptor density revealed by functional

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characterization of the human FSH receptor A189 V mutation. Mol. Cell. Endocrinol. 331, 109-

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Uchida, S., Uchida, H., Maruyama, T., Kajitani, T., Oda, H., Miyazaki, K., Kagami, M.,

733

Yoshimura, Y., 2013. Molecular analysis of a mutated FSH receptor detected in a patient with

734

spontaneous

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hyperstimulation

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syndrome.

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Dias, J.A., Ulloa-Aguirre, A., 2008. Functional and structural roles of conserved cysteine

739

residues in the carboxyl-terminal domain of the follicle-stimulating hormone receptor in human

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embryonic kidney 293 cells. Biol. Reprod. 78,869-882. doi: 10.1095/biolreprod.107.063925.

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Vassart, G., Pardo, L., Costagliola, S., 2004. A molecular dissection of the glycoprotein hormone

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Savagner, F., Croué, A., Mathieu, E., Lahlou, N., Descamps, P., Misrahi, M., 2003. A chorionic

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748

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Zariñán, T., Perez-Solís, M.A., Maya-Núñez, G., Casas-González, P., Conn, P.M., Dias, J.A.,

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Ulloa-Aguirre, A., 2010. Dominant negative effects of human follicle-stimulating hormone

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receptor expression-deficient mutants on wild-type receptor cell surface expression. Rescue of

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oligomerization-dependent defective receptor expression by using cognate decoys. Mol. Cell.

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Endocrinol. 321, 112-122. doi: 10.1016/j.mce.2010.02.027.

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Figure legends:

761

Fig 1: 3-D representation of the X-ray crystal structure of FSH-FSHRED complex (PDB ID:

763

4AY9)

764

The leucine rich repeats of FSHRED are shown in pink, whereas the green and dark blue colours

765

represent FSH α- and β- subunits respectively. L2β loop of FSH is shown in cyan and the

766

residues chosen for mutagenesis studies are indicated by yellow colour. Inset shows the positions

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of the residues K146, E197, V221, I222 and K243 of FSHRED.

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Fig 2: FSHR expression in WT and substitution point mutants of FSHR by Western blot

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Cell lysates from CHO cells transiently expressing wild type FSHR (WT) and the substitution

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point mutants were subjected to 7.5% SDS-PAGE. The blot was probed with anti-FSHR

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772

antibody (upper panels) and anti-beta-actin antibody (lower panels). Equal beta actin levels

773

across all lanes ensured equal protein loading. Image shown is a representative one.

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Fig 3: Visualization of cell surface expression of FSHR in WT and substitution point

776

mutants by confocal microscopy

777

Using a monoclonal antibody against the FSHR conjugated to an Alexa-488 fluor dye, direct

778

immunofluorescence experiments were carried out. FSHR expression can be visualized as green

779

staining in CHO cells transiently expressing the WT/mutants of FSHR. CHO cells transiently

780

transfected with empty vector pcDNA 3.1+ which did not encode FSHR served as a negative

781

control. DAPI (blue) was used as a nuclear stain. Images shown are representative of three

782

independent experiments. Scale bar =20 μm.

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Fig 4: Quantitation of cell surface FSHR expression of WT and substitution point mutants

785

of FSHR by flow cytometry

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The cell surface FSHR expression in CHO cells transiently expressing wild type FSHR (WT)

787

and the substitution point mutants using anti-FSHR antibody conjugated to Alexa-488 fluor dye.

788

The mean fluorescence intensity for the mutants was plotted by comparing the values to that

789

obtained for the WT receptor which was considered to be one. Data represents mean ± SEM of

790

three independent experiments carried out in duplicates. No significant differences were

791

observed.

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Fig 5: Radioreceptor assay to estimate the amount of surface bound and internalized FSH-

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FSHR complexes

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(A)The hormone binding ability of WT/mutant FSHR constructs was determined by incubating

796

CHO cells transiently expressing WT/mutant receptor constructs with labeled FSH (125I-FSH) in

797

the presence of saturating concentration of unlabeled FSH. (B)The ratio of counts of the

798

internalized FSH-FSHR complexes to the sum of the surface bound and internalized counts (total

799

counts) has been expressed as a percentage for the WT/mutant FSH receptors. The graphs

800

represents mean ± SEM of three independent experiments performed in duplicates. Statistical

801

analysis was performed using the unpaired student’s t-test. *P < 0.05, **P < 0.01 with respect to

802

WT FSHR (for both the surface bound counts and internalized counts) represents statistical

803

significance.

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Fig 6: Dose response displacement binding curve with 125I-FSH

806

Binding of FSH to WT/substitution point mutants of FSHR was evaluated in CHO cells

807

transiently expressing the receptor constructs. Forty eight hours post transfection, cells were

808

incubated with

809

assay was performed thrice and the graph represents mean ± SEM of a single representative

810

experiment performed in duplicates.

I-FSH in the presence of increasing concentrations of unlabeled FSH. The

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Figure 7: Estimation of FSH induced cAMP production in WT and substitution point

813

mutants of FSHR by cAMP enzyme immunoassay

814

Forty eight hours post transfection of CHO cells expressing WT FSHR or its point mutants, the

815

cells were either unstimulated (basal cAMP response) or stimulated with increasing doses of

816

FSH: 1ng, 10 ng and 100 ng of FSH for one hour at 37ºC. Cell lysates prepared subsequently

817

were subjected to cAMP enzyme immunoassay and the response obtained by WT FSHR or the

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point mutants were determined and plotted. The graph represents mean ± SEM of three

819

independent experiments performed in duplicates. Statistical analysis was performed using the

820

unpaired student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001 with respect to WT FSHR

821

represents statistical significance.

822

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Fig 8: cAMP production in WT and substitution point mutants of FSHR post stimulation

824

with the glycoprotein hormones hCG, TSH and LH

825

Determination of basal cAMP response (0) and in response to stimulation with (A) 10 ng, 100

826

ng, 500 ng and 1000 ng hCG (B) 10 ng and 100 ng of TSH and (C) 10 ng and 100 ng of LH in

827

CHO cells transiently expressing WT and mutant FSHR constructs and incubated with the

828

hormones mentioned above for one hour at 37 ºC. Cell lysates prepared subsequently were

829

subjected to cAMP enzyme immunoassay and the response obtained by WT FSHR or the point

830

mutants were determined and plotted. The graph represents mean ± SEM of three independent

831

experiments performed in duplicates. Statistical analysis was performed using the unpaired

832

student’s t-test. *P < 0.05 with respect to WT FSHR represents statistical significance.

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Table 1 FSHR residues selected for mutational analysis

FSHR mutant

Rationale for selection of the residue

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generated Naturally occurring mutations V221G

Reported in a patient with primary amenorrhea (Nakamura et al., 2008)

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To mimic the heterozygous condition observed in the patient, co-transfection was carried out with WT and V221G construct (represented as WT/V221G) Reported in a patient with sOHSS (Chauhan et al., 2015)

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T449N

To mimic the heterozygous condition observed in the patient, co-transfection was carried out with WT and T449N construct (represented as WT/ T449N) Induced mutations K146N

Predicted to be involved in interaction with FSH based on in silico analysis

E197S

TE D

(Núñez Miguel et al., 2008)

These residues are present at the FSH-FSHRED interaction

I222T

interface as indicated by X-ray crystal structure analysis (Jiang et al., 2014)

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K243R

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Table 2

Observations

of

Protein

Interactions

Calculator

(PIC)

analysis

indicating

the

hydrophobic/ionic/main chain-main chain and main chain-side chain interactions between WT

Residues involved in the interaction

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FSHR and residues in beta subunit of FSH.

Type of interaction

FSH β P42 A43 A43

E197

R44

K243

A43

Main chain-side chain

E197

R44

Side chain-side chain

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Hydrophobic

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WT FSHR V221 V221 I222

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Specific binding of 125I-FSH (cpm)

EC50 (ng)

cAMP assay

2.56±0.06 2.45±0.02 2.43±0.01 3.00±0.02 2.31±0.03 2.25±0.02 2.17±0.01 2.27±0.02 2.38±0.03 2.67±0.01

3106±44 3089±53 2109±70 1972±69 2002±35 1019±42 2130±48 3093±40 3133±35 3044±46

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62974±2751 59056±4571 18618±1097 20586±1321 35615±2157 23767±6107 33318±3196 106080±3466 91802±2420 79882±3929

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Emaxat 100 ng FSH (pmol/well)

TE D

100 92±19 83±30 90±12 93±21 100±10 97±5 104±16 114±11 102±13

Radioreceptor assay

EP

WT K146N E197S V221G WT/V221G V221K I222T K243R T449N WT/T449N

Cell surface receptor expression by flow cytometry (% of WT)

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FSHR construct

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Functional characteristics of cell surface FSHR expression by flow cytometry, radioreceptor assay: specific binding of 125I-FSH (cpm) and EC50 values (concentration of unlabeled FSH needed for 50% displacement), Emax at 100 ng FSH (maximal response) and EC50 (FSH required for attaining 50% of Emax) of FSH-induced cAMP production by WT and mutant FSH receptors. Values represent mean ± SEM from three independent experiments performed in duplicates.

EC50 (ng) 8.27±0.20 8.05±0.04 17.20±0.35 16.04±1.02 11.47±0.67 38.17±2.94 8.96±0.39 3.12±0.054 3.31±0.18 2.78±0.20

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Conflict of Interest

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