Báo cáo khoa học: Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor pptx

Double mutant receptors containing two activating TSHr mutations harboring the amino acid substitution P639S 6th transmembrane segment [17] and I486M 1st extracellular loop [3], named I4

Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor

Patrizia Agretti1, Giuseppina De Marco1, Paola Collecchi2, Luca Chiovato3, Paolo Vitti1, Aldo Pinchera1 and Massimo Tonacchera1

1

Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro, Universita` di Pisa, Pisa, Italy;

2

Dipartimento di Oncologia, Divisione di Anatomia Patologica, Universita` di Pisa, Pisa, Italy;3Cattedra di Endocrinologia, Fondazione S Maugeri IRCCS, Pavia, Italy

Activating mutations of the thyroid-stimulating hormone

receptor (TSHr) have been identified as a cause of toxic

adenomas Germline-inactivating TSHr mutations have

been described as a cause of congenital hypothyroidism The

effects of combining activating and inactivating mutations

within a single receptor was studied The double mutant

T477I/P639S contained an activating TSHr mutation

(P639S) together with an inactivating one (T477I) The other

one (I486M/P639S) contained two activating mutations

Constructs were expressed in COS-7 cells and basal and

TSH-stimulated cyclic AMP (cAMP) accumulation and

inositol phosphate (IP) production were determined The

expression at the cell surface was studied both with binding

and fluorescence-activated cell scanning analysis Our results

show that the effect of combining the two activating

muta-tions is an increase in the constitutive activity only for the cAMP pathway and not for the IP pathway suggesting that different mutations result in receptor conformations with different relative abilities to couple to Gs-alpha or Gq-alpha Surprisingly the double mutant containing the T477I behaves as an activating receptor with constitutive activity both for the cAMP and IP pathways These data show that

an inactive form of the TSHr which is trapped inside a cell after transfection is able to gain the membrane surface when combined with an activated form of the receptor

Keywords: TSH receptor; G-protein-coupled receptors; constitutive activity; site-directed mutagenesis; somatic mutations; germline mutations

G-protein-coupled, seven transmembrane segment

recep-tors comprise the largest superfamily of proteins in the body

[1] Many G-protein coupled receptors have a certain basal

activity (constitutive activity) and thus can activate

G-proteins in the absence of the agonist [1,2] Interestingly,

it has been encountered that discrete mutations of these

receptors are able to dramatically increase this constitutive

agonist-independent receptor activity [3] The

thyroid-stimulating hormone receptor (TSHr), together with the

follicle-stimulating hormone (FSH) and the luteinizing

hormone (LH) receptors, is a member of a subfamily of

seven transmembrane G-protein-coupled receptors,

charac-terized by a large N-terminal extracellular domain involved

in hormone binding [4,5]; the receptor is mainly coupled to

adenylyl cyclase via Gs-alpha and, in some species including

man, it activates also the inositol phosphate cascade (IP) via

a Gq-alpha protein [6–8] Current models of G-protein-coupled receptor activation consider that binding of the ligand, within the slit formed by the transmembrane helices (for biogenic amines), and/or to the extracellular loops (for peptide ligands), relieves a built-in negative constraint by stabilizing an active conformation of the receptor [9] In this conformation, the new position of the transmembrane helices translates into an increased affinity of the intracel-lular loops for G-proteins Somatic and germline activating mutations of the TSHr gene have been identified as a major cause of toxic thyroid adenoma [3,10,11] and hereditary or sporadic nonautoimmune toxic thyroid hyperplasia [12,13], respectively On the contrary, inactivating mutations aboli-shing basal activity or affecting agonist induced response have been described in cases of congenital hypothyroidism with thyroid hypoplasia [14–16]

All activating TSHr mutations have been shown to activate adenylyl cyclase when expressed in eukaryotic cells [3,10] Some of these mutations possess also constitutive activity for the IP pathway [3,11] Inactivating mutations of the TSHr gene may be due to truncated forms of the receptor

or to point mutations [14–16] It has been demonstrated

in vitro that point mutations can alter the routing of the receptor to the cell surface, resulting in loss of basal activity and loss of agonist induced cAMP production [14–16]

In order to study the mechanism of activation of the TSHr we explored the effects of combining previously

Correspondence to M Tonacchera, Dipartimento di Endocrinologia,

Via Paradisa 2, 56124 Pisa, Italy Fax: 050 578772, Tel.: 050 995048,

E-mail: mtonacchera@hotmail.com

Abbreviations: bTSH, bovine TSH; FACS, fluorescence-activated

cell scanning; FSH, follicle stimulating hormone; G q -alpha,

G-protein q alpha; G s -alpha, G-protein s alpha; IP, inositol

phosphate; KRH, Krebs/Ringer/Hepes; LH, luteinizing hormone;

TSH, thyroid-stimulating hormone.

(Received 11 July 2003, accepted 1 August 2003)

described mutations within a single receptor We decided to

combine two particularly potent activating TSHr mutations

and to combine an activating mutation together with an

inactivating one

Double mutant receptors (containing two activating TSHr

mutations) harboring the amino acid substitution P639S (6th

transmembrane segment) [17] and I486M (1st extracellular

loop) [3], named I486M/P639S, and the double mutant

receptor (containing an activating together with an

inacti-vating TSHr mutation) P639S and T477I (1st extracellular

loop) [16], named T477I/P639S, were constructed

Con-structs were subcloned in the expression vector pSVL and,

after transient expression in COS-7 cells, basal and

TSH-induced cAMP and IP production were determined

Cell-surface expression was evaluated with [125I]bTSH (bovine

TSH) binding, an enzyme immunosorbent assay (EIA) and a

fluorescence-activated cell scanning (FACS) analysis using

different monoclonal antibodies against the TSHr

Our results show that the effect of combining the two

activating mutations is an increase in the constitutive

activity only for the cAMP pathway and not for the IP

pathway suggesting that different mutations result in

receptor conformations with different relative abilities to

couple to Gs-alpha or Gq-alpha Surprisingly the double

mutant containing the T477I behaves as an activating

receptor with constitutive activity both for the cAMP and

IP pathways These data show that an inactive form of the

TSHr which is trapped inside a cell after transfection is able

to gain the membrane surface when combined with an

activated form of the receptor

Materials and methods

Construction of mutant TSH receptors

The constructs harboring the single mutated TSH receptors

T477I, I486M and P639S have been described [11,16,17] In

brief, a SpeI-CvnI segment (1660–1932) or a CvnI-BstEII

segment (1932–2450) in the cDNA of the wild type (WT)

TSHr in the expression vector pSVL, was replaced by a

homologous segment harboring the mutation in position

477 and 486 or 639, respectively These mutated sequences

were cloned directly from DNA extracted from nodular

tissues obtained from patients with toxic multinodular

goiter (I486M, P639S) or from the blood of a patient with

a germline inactivating TSHr mutation (T477I) The SpeI

restriction site in the WT-TSHr was created by site-directed

mutagenesis (the change in the coding region does not

modify the encoded amino acid sequence)

For the construction of the double mutants, a

CvnI-BstEII fragment in the T477I and I486M TSHr was

substituted by homologous segments harboring the P639S

mutation, yielding the T477I/P639S or I486M/P639S

dou-ble mutant, respectively (Fig 1) The resulting constructs

were sequenced directly by an ABI PRISM 310 Genetic

Analyzer (PE Applied Biosystems, Foster City, CA, USA)

to verify the presence of the mutations

Expression in eukaryotic cells of mutated genes

For transient expression, COS-7 cells were seeded at the

concentration of about 150 000 cells per 3-cm dish for

binding, EIA and FACS analysis, cAMP and IP determination COS-7 cells were grown in DMEM sup-plemented with 10% fetal bovine serum, penicillin

100 IUÆmL)1, streptomycin 100 lgÆmL)1, fungizone 2.5 lgÆmL)1 and 1 mM sodium pyruvate One day after seeding, cells were transfected with the DEAE–dextran method followed by a 2-min 10% dimethylsulfoxide shock [18]

For functional assays, 48 h after transfection cells were used for cAMP or IP determinations and for EIA, FACS analysis and [125I]bTSH binding studies Triplicate dishes were used for each condition and each experiment was repeated at least three times Results were expressed as mean ± SEM from one representative experiment When not shown, SEM values were so small that they fall within the symbols

cAMP assay Cells were washed with Krebs/Ringer/Hepes buffer (KRH) and preincubated for 30 min at 37C This was followed

by a 1-h incubation at 37C in the presence of 0.5 mM isobutylmethyl xanthine as a cAMP phosphodiesterase inhibitor, in the absence of bTSH (basal values), or in the presence of various concentrations of bTSH (Sigma Chemical Co) At the end of the incubation the medium was removed and replaced by 0.1MHCl The cell extracts were dried in a vacuum concentrator and cAMP was determined as described [17] and expressed as picomoles per dish

Fig 1 Schematic representation of the double mutants TSHr (I486M/ P639S; T477I/P639S).

Inositol phosphate assay

For IP determinations, 24 h after transfection cells were

incubated with 20 lCiÆmL)1[3H]inositol (Amersham

Phar-macia Biotech Europe, Germany) The next day, dishes

were washed three times with KRH, preincubated in KRH

plus LiCl 10 mM for 30 min at 37C and incubated for

18 min at 37C in the presence of KRH plus LiCl 10 mM

(basal values) or in the same medium plus different

concentrations of bTSH The incubation was stopped by

addition of ice cold 3% HClO4, and3H-labeled IP were

isolated and assayed by stepwise chromatography on

AG1· 8 resin [19] The cell debris in the bottom of the

dishes was dissolved in 1MNaOH and counted as

phos-phatidyl inositols Results were expressed as percentage

radioactivity incorporated in inositol phosphates (IP1+

IP2+ IP3) over the sum of radioactivity in inositol

phosphates and phosphatidyl inositols [19]

Binding assay

Forty-eight hours after transfection, cells were washed once

with Hanks’ solution in which NaCl was replaced by

sucrose 280 mM containing 0.2% bovine serum albumin

(BSA) and 2.5% low fat milk Binding studies were

performed by incubating cells in that same medium at

room temperature for 4 h in the presence of about

90 000 c.p.m [125I]bTSH (a gift of BRAHMS, Berlin,

Germany) and the appropriate concentrations of cold

bTSH At the end of the incubation cells were rinsed twice

with ice cold Hanks’ medium, solubilized with 1MNaOH

and bound radioactivity was determined in a

gamma-counter In the absence of a consensus about the bioactivity

of pure bovine TSH [20], we have expressed all TSH or

TSHr concentrations in mUÆmL)1, assuming a 1/1

stechio-metry for TSH binding to its receptor The competition

binding curves have been fitted by nonlinear regression

assuming a single receptor site [21]

Enzyme immunosorbent assay (EIA)

EIA measurements were carried out with transfected cells,

nonpermeabilized and in suspension Forty-eight hours

after transfection cells were detached from the plates with

NaCl/Picontaining 5 mM EDTA and EGTA Cells were

then pelleted and incubated with a mouse anti-(human

TSHr Ig) (Novocastra Laboratories Ltd, UK) diluted at

1 lgÆmL)1 in NaCl/Pi containing 0.5% BSA After two

washes with NaCl/Pi, cells were incubated for 1 h at 4C

with peroxidase-conjugated anti-(mouse IgG) as secondary

antibody (Sigma Chemical Co.) diluted 1 : 70 000 in NaCl/

Picontaining 0.5% BSA The cells were washed three times

with NaCl/Piand, finally, incubated with the

o-phenylene-diamine dihydrochloride substrate (Sigma Chemical Co.)

for 30 min at 37C The reaction was stopped by adding

1M H2SO4 and color development was measured at

492 nm

FACS analysis

Cells were detached from culture dishes with 5 mmolÆL)1

each of ethylenediamine tetraacetate and

ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N¢,N¢-tetraacetic acid in NaCl/Pi and transferred to Falcon tubes (2052, Falcon Labware, Cockeysville, MD, USA) Cells were washed with NaCl/Piplus 0.1% BSA, centrifuged at 500 g at 4C for

3 min, and treated appropriately for the nonpermeabilized

or permeabilized cell assay as described previously [16] Nonpermeabilized cells were incubated at room tempera-ture for 30 min with 200 lL of a monoclonal antibody directed against the TSHr (BA8 gently gifted from Dr Sabine Costagliola) diluted in NaCl/Piplus 0.1% BSA A blank sample was prepared by incubating cells with 200 lL NaCl/Piplus 0.1% BSA For the permeabilized cell assay, cells were fixed with 2% NaCl/Pi/paraformaldehyde (UCB, Brussels, Belgium) and then treated for 30 min with NaCl/Pi plus 0.1% BSA and 0.2% saponin (Sigma Chemical Co.,

St Louis, MO, USA); all subsequent steps with antibodies were performed in 0.2% saponin Cell-bound monoclonal antibodies were detected washing the cells with NaCl/Piplus 0.1% BSA and then incubating them for 30 min at 4C in the dark with a goat anti-(mouse IgG) fluorescin-conjugated (Becton Dickinson and Co., San Jose, CA, USA) diluted

1 : 20 in NaCl/Pi with 0.1% BSA containing 10 lgÆmL)1 propidium iodide (Sigma) Propidium iodide (PI) was used

to detect and exclude from the analysis damaged cells Flow cytometric analysis was performed using a FACSort flow cytometer (Becton Dickinson and Co.) equipped with a laser for an excitation at 488 nm to detect monoclonal antibodies conjugated with fluorescin 5-isothiocyanate and

PI Fluorescence emission of fluorescin 5-isothiocyanate and PI from single cells were separated and measured using the standard optics of the FACSort The CELLQUEST software program (Becton Dickinson and Co.) was used

to acquire and analyze data A minimum of at least 10 000 cells was analyzed

Computation of specific constitutive activity (SCA) and relative SCA (RSCA)

Given that the transfection efficiency for each construct is constant for a given batch of cells, the SCA was calculated by:

SCA¼ ðAr  AvÞ=ðFr  FvÞ where Ar and Av are the cAMP (or IP) of cells transfected with the mutant constructs and vector, respectively, and Fr and Fv are the corresponding mean fluorescence unit obtained with FACS analysis RSCA, which is normalized

to the WT-TSHr, was obtained by:

Results

cAMP cascade The effects of each TSH receptor construct harboring one

or two mutations have been investigated after transient expression in COS-7 cells

For basal cAMP determinations, 250 ng per dish of the DNA of the various constructs, giving maximal cAMP stimulation [3], were used to transfect COS-7 cells As expected, cells transfected with the WT-TSHr exhibited a

threefold increase in basal cyclic AMP accumulation with

respect to cells transfected with vector alone, showing

constitutive activity (Table 1, Fig 2A) Cells transfected

with two of the constructs harboring a single mutation

(I486M; P639S) displayed higher level of basal cAMP as

compared to cells transfected with the WT-TSHr (Table 1,

Fig 2A), while cells transfected with the construct

harbor-ing the T477I mutation showed levels of cAMP that were

very similar to those exhibited from the cells transfected

with the empty vector alone The I486M/P639S double

mutant showed a further increase of cAMP accumulation

with respect to the single parental mutated receptors

Surprisingly the combination of the activating P639S with

the inactivating T477I yielded a receptor T477I/P639S

with a strong constitutive activity similar to that obtained

with P639S

The biological response to bTSH of the cells transfected

with the DNA of the different constructs was explored in

terms of cAMP accumulation (Table 1, Fig 2A) Cells

transfected with the constructs harboring a single activating

mutation (P639S; I486M) exhibited a maximal stimulation

to bTSH that was similar to that observed with the WT-TSHr Cells transfected with the inactivating mutation T477I showed a very low response to bTSH with values of cAMP production at 100 mUÆmL)1of bTSH about seven-fold lower than the WT-TSHr The I486M/P639S double mutant showed an increase of maximal cAMP accumula-tion after bTSH challenge with respect to the single mutated receptors The T477I/P639S maintained a cAMP response

to bTSH similar to P639S alone

Inositol phosphate cascade

As expected from previous studies [3,11], no significant increase of basal levels of IP production was observed in the cells transfected with the WT-TSHr (Table 1, Fig 2B); a slight increase over values obtained with an empty vector or the wild-type receptor construct, was observed in cells transfected with the P639S or I486M single mutant, showing constitutive activity for the IP production (Table 1,

Table 1 Functional characteristics of TSHr mutants transfected in COS-7 cells as described in materials and methods cAMP values, expressed as percentage basal cAMP levels of WT-TSHr, were measured in basal conditions and after stimulation with 10 and 100 mUÆmL)1bTSH IP values, expressed as percentage basal IP levels of WT-TSHr, were measured in basal conditions and after stimulation with 100 mUÆmL)1bTSH Results are mean ± SEM of three independent experiments.

Fig 2 Basal and bTSH-induced stimulation of cAMP and IP levels in COS-7 cells transfected with the single or double mutated receptors Values are mean ± SEM from one representative experiment in which triplicate dishes were used (A) Basal intracellular cAMP values (picomoles per dish) and levels of cAMP production after stimulation with 10 and 100 mUÆmL)1bTSH in COS-7 cells transfected with saturating concentrations of DNA (250 ng per dish), of single mutated receptors (T477I, I486M, P639S) or double mutant receptors (T477I/P639S; I486M/P639S), WT-TSHr and pSVL alone, are shown (B) Basal IP values, expressed as percentage of (IP + PI) and IP levels after bTSH stimulation (100 mUÆmL)1) in COS-7 cells transfected with saturating concentrations of DNA (250 ng per dish), of single mutated receptors (T477I, I486M, P639S) or double mutant receptors (T477I/P639S; I486M/P639S), WT-TSHr and pSVL alone, are shown.

Fig 2B) Cells transfected with the T477I mutation showed

basal IP values similar to those obtained with pSVL or

WT-TSHr alone Cells transfected with the I486M/P639S double

mutant showed basal IP values intermediate to those

obtained in cells transfected with the single parental

mutants, showing that the double mutant promoted the

same higher basal IP values Cells transfected with the

T477I/P639S double mutant showed only a slight increase

in basal IP production with respect to WT-TSHr or T477I

alone, and a clear decrease with respect to P639S

Stimulation of IP accumulation by 100 mUÆmL)1 of

bTSH exhibited a threefold stimulation of IP production in

cells transfected with the WT-TSHr (Table 1, Fig 2B) The

I486M and P639S mutated receptors showed an increased

production of IP with respect to the WT-TSHr after bTSH

stimulation The T477I completely lost the ability to

respond to bTSH The I486M/P639S maintained a similar

production of IP after bTSH challenge with respect to the

parental TSH receptors The T477I/P639S was able to

produce a significant IP stimulation after bTSH challenge,

which was lower to that produced by P639S alone

Binding of [125I]bTSH

To measure the total number of receptors (or TSH binding

capacity, Bmax) expressed at the surface of the cells

transfected with the different constructs, and their relative

dissociation constants (Kd), binding studies were performed

with a bovine [125I]TSH tracer as described in Materials

and methods

Cells transfected with the constructs harboring the single

activating mutations I486M and P639S, exhibited a lower

level of expression as compared to the WT-TSHr, indicating

that the increased cAMP production (measured in the same

experiment) was not due to overexpression of the mutated

receptors (Fig 3) When compared with the WT-TSHr, the

level of receptor expression in COS-7 cells transfected with

the T477I inactivating mutant was about four times lower

(Fig 3) The I486M/P639S double mutant showed a similar

level of expression with respect to the single mutated

receptors Surprisingly, also the T477I/P639S showed a

good ability to be expressed at the surface of the cells As

already noted earlier [3], constitutively active receptors

showed a higher affinity for bTSH binding than the

WT-TSHr (Fig 3)

EIA and FACS analysis

The level of receptor expression on the cell surface for the

different constructs was independently measured by EIA,

using a monoclonal antibody directed against the

extracel-lular domain of the TSH receptor (NCL-TSH-R2,

Novo-castra Laboratories Ltd, Newcastle, UK) (data not shown),

and by FACS analysis using BA8, a monoclonal antibody

directed against the TSHr (Fig 4) COS-7 cells transfected

with the inactivating mutant T477I showed a very low level

of expression of the mutated receptor at the cell surface

(Fig 4A) The T477I was however, clearly detectable within

the cells after saponin permeabilization (Fig 4B),

suggest-ing that the T477I receptor was synthesized and recognized

by the antibody, but might have a defect in folding of the

mutant protein resulting in abnormal routing to the plasma

membrane The level of expression at the cell surface of the single mutated receptors (I486M, P639S) and the I486M/ P639S double mutant was similar and was the same according to the binding experiments (Table 2) The FACS analysis also confirmed that the T477I/P639S double mutant was able to reach the membrane surface The comparison of WT-TSHr and mutated receptors expression

at cell surface by using the three methods are shown in Table 2 The level of receptor expression on the cell surface was statistically different for all the single and double mutant receptors with respect to the WT-TSHr The Student’s t-test with 95% confidence was used to establish significance (P£ 0.05)

Fig 3 Binding characteristics of the single and double mutants expressed in COS-7 cells [125]IbTSH binding to COS-7 cells transfected with 250 ng per dish of the construct harboring the single mutated receptors (T477I, I486M, P639S) or double mutant receptors (T477I/ P639S; I486M/P639S) and WT-TSHr The B max , total receptor amount (binding capacity; expressed as milliunits of TSH per milliliter) and K d (also ex pressed in mUÆmL)1of TSH) were computed as des-cribed in Materials and methods (the SEM values are so small that they fall within the symbols).

Specific constitutive activity

The data obtained from the FACS analysis allowed

measurement of efficiency of transfection and computation

of the increase in cAMP and IP within the effectively

transfected cells Assuming that the mutations did not affect

recognition by the monoclonal antibodies, these data also allowed normalization of the increase in cAMP and IP accumulation to the amount of receptor expressed at the cell surface, yielding an estimation of specific constitutive activity When transfecting COS-7 cells with increasing amounts of wild-type and mutant cDNAs we observed that the constitutive activity of the WT and mutated receptors was linear over the range of cell surface expression (data not shown) The I486M showed to be eightfold and the P639S 10-fold more active than the WT-TSHr The combination

of the two activating TSHr mutations produced a I486M/ P639S double mutant that showed an increase of SCA with respect to the parental constructs (11-fold with respect to the WT-TSHr) (Fig 5A) The combination of the activating/ inactivating T477I/P639S double mutant produced a recep-tor that was 7.5 more active than the WT-TSHr but less active with respect to the P639S alone

These data were also used to compute IP accumulation with respect to the amount of receptor expressed at the cell surface The data clearly showed that both the single activated TSHr receptors had constitutive activity with respect to the WT-TSHr (Fig 5B) being 15-fold for the I486M and 33-fold for the P639S The combination of the two activating TSHr mutations produced a receptor which

Fig 4 Expression analysis of the single and double mutants by FACS analysis using the BA8 monoclonal antibody Fluorescence intensity is expressed

in arbitrary units, as a function of cell number plotted on a logarithmic scale (A) Nonpermeabilized cells assayed after transfection with pSVL, TSHr WT, the T477I, I486M and P639S single mutants, and the T477I/P639S and I486M/P639S double mutants BA8 immunoreactivity (arbitrary units): vector, 3.20; TSHr WT, 19.67; T477I, 5.04; I486M, 15.00; P639S, 14.20; T477I/P639S, 15.52; I486M/P639S, 16.21 (B) Saponin-permeabilized cells identically transfected BA8 immunoreactivity (arbitrary units): vector, 6.00; TSHr WT 11.80; T477I, 15.13; I486M, 17.70; P639S, 22.37; T477I/P639S, 20.16; I486M/P639S, 17.68.

Table 2 The level of receptor expression on the cell surface for the

different constructs after transfection was measured by [125I]bTSH

binding, EIA and FACS analysis Data are represented as a percentage

of the values corresponding to WT-TSHr minus the nonspecific

absorbance readings for EIA, the specific binding and the

non-specific fluorescence for FACS analysis The nonnon-specific data were

obtained transfecting COS-7 cells with the empty vector Values are

mean ± SEM of three independent experiments.

[ 125 I]bTSH binding EIA FACS

T477I/P639S 77 ± 10.8 67 ± 7.1 78 ± 12.1

I486M/P639S 77 ± 9.5 72 ± 9.5 81 ± 8.8

was less potent that the P639S alone (24-fold over the

WT-TSHr) while the combination of the

activating/inacti-vating T477I/P639S produced a constitutive activity of

sixfold with respect to the WT-TSHr

Discussion

According to the extension of the ternary complex model

[22] for activation of G-proteins by serpentine receptors, the

receptor is assumed to exist in two interconvertible

confor-mations: the inactive one, enforced by a built-in negative

constraint, corresponds to the major unliganded form of the

receptor; the active conformation capable of promoting

GDP/GTP exchange on the G-protein is achieved by a

fraction of the unliganded receptors only, but is stabilized by

binding of its ligand The model predicts that unliganded

receptors may display a basal activity (corresponding to the

fraction of active receptors) which can be antagonized by

inverse agonists, the effect of which is to stabilize the

inactive conformation [22] The TSH receptor offers a series

of characteristics which make it an interesting model to

study in this context: (a) it is capable of activating both

Gs-alpha and Gq-alpha [6–8]; (b) it displays an easily

measurable constitutive activity towards adenylyl cyclase

after transfection in COS cells [1,3,10,11]; (c) it can be

activated by a surprisingly diverse spectrum of point

mutations, or (d) by stimulating autoantibodies; (e)

loss-of-function mutations have also been described, abolishing

basal activity or affecting agonist-induced response and (f) the mechanism by which binding of the hormone to the N-terminal domain of the receptor translates into activation

of its serpentine domain is unknown

With the aim of improving our understanding of the mechanisms underlying some of these characteristics, we have explored the effects of combining activating and inactivating mutations of the TSHr within a single receptor

on its constitutive activity towards Gs-alpha and Gq-alpha and its binding characteristics and responsiveness to bTSH When two strong activating mutations of the TSHr gene (both possessing constitutive activity for both cAMP and IP pathways) have been combined a further increase of basal and bTSH-stimulated cAMP production was observed with respect to the single parental receptors The expression of the I486M/P639S on the cell surface was similar to the single mutated receptors These results suggest that the double mutant I486M/P639S may be one step further in a scale of constraint release and the two mutations cooperate to potentiate the activity of the receptor The increase of basal activity by combining activating mutations is well known for glycoprotein hormone receptors [23,24] No further increase of IP was evident in the double mutant I486M/ P639S with respect to the parental receptors This observa-tion suggests the possible existence of multiple conformaobserva-tion states with different abilities to interact with Gs-alpha and or

Gq-alpha The mutational dissociation of functional con-formations have also been described for glycoprotein hormone receptors [25–27] The T477I has been described

as an inactivating mutation in a patient with congenital hypothyroidism and thyroid hypoplasia [16] Similarly, an inactivating mutation of the FSH receptor gene that is located in the close vicinity of the amino acid 477 of the TSHr was found in a patient affected by primary ovarian failure [28] When the T477I was transiently expressed in COS-7 cells we observed the complete loss of basal constitutive activity for the cAMP pathway, a strong reduction in bTSH stimulated cAMP production and absent bTSH stimulated IP production The level of receptor expression in COS-7 cells transfected with the T477I measured both by binding with bTSH and by using a monoclonal antibody in FACS analysis directed against the TSHr was very low The very low expression of the mutated receptor on the cell membrane is probably due to the poor routing of the receptor at the cell surface Surprisingly the effect of combining this T477I with the activating P639S produced a double mutant with a similar level of expression

at the cell surface with respect to the parental P639S The T477I/P639S behaves like the P639S alone, in terms of basal and bTSH stimulated cAMP accumulation Only a slight decrease in the basal and bTSH stimulated IP production was noticed transfecting this T477I/P639S with respect to P639S alone

These data clearly show that an inactivated G protein-coupled receptor was trapped intracellularly after transient expression in eukaryotic cells and when combined with an activated form of the receptor reconstituted a functional membrane receptor Similarly, a D578H mutant LH receptor was shown to increase the cell surface expression

of poorly expressed vasopressin-LH receptor’s chimera [29] However, contrary to what has been observed in our model where the P639S has a lower expression at the cell surface

Fig 5 RSCA of single and double mutant receptors for cAMP (A) and

IP (B) computed as described in Materials and methods Results are the

mean ± SEM from one representative experiment in which triplicate

dishes were used.

with respect to the WT-TSHr, the D578H mutant alone was

shown to be expressed at the cell surface four times more

than the WT-LH receptor [29] Maya-Nunez et al [30]

showed that a mutant (E90K) gonadotropin-releasing

hormone receptor (GnRHr) was rescued either by deleting

K191 or by adding a C-terminal sequence Either of these

approaches alone supported high-membrane expression of

the GnRHr It has been proposed that the E90K mutation

itself while resulting in protein misfolding does not

irreversibly destroy the intrinsic ability of the mutant to

bind ligand or to couple effectors The rescued receptor,

now stabilized in the plasma membrane, was able to activate

the appropriate effector system The proper folding and

assembly of polypeptides occur in the endoplasmic

reti-culum If polypeptides cannot fold correctly, mechanisms

of quality control ensure that the aberrant protein is not

further processed along the secretory pathway [31] The

quality control mechanisms are mediated by a family of

proteins called molecular chaperones Recently, a class of

compounds called chemical chaperones were shown to

reverse the intracellular retention of several misfolded

proteins [31] Besides it has been described that small

cell-permeable molecules can act as either chemical chaperones

or pharmacological (ligand-mediated) chaperones to rescue

mutant protein function [31] Thus, the development of

strategies aimed at promoting proper folding and

matur-ation of mutant proteins could provide new therapies for a

wide spectrum of diseases [31]

In conclusion, the combination of two activating

muta-tions of the TSHr determined an increase in the activity only

for the cAMP pathway and not for the IP pathway

suggesting that different activating mutations result in

receptor conformations with different relative abilities to

stimulate the cAMP or IP regulatory cascades Surprisingly

the combination of a strong inactivating mutation with an

activating one produced a receptor that was able to be

expressed at the cell surface with high constitutive activity

Acknowledgements

This work was supported by the following grants: Ministero

dell’Uni-versita` e della Ricerca Scientifica (MURST), Programma di Ricerca: Le

malattie della tiroide: dalle basi molecolari alla clinica Ministero

dell’Universita` e della Ricerca Scientifica (MURST), Programma di

Ricerca: Strategie per la valutazione degli effetti disruptivi dei

contaminanti ambientali sul sistema endocrino degli animali e

dell’uomo CNR Progetto Biotecnologie CTB 99.00.224.PF 31: Basi

molecolari delle neoplasie benigne e maligne della tiroide Istituto

Superiore di Sanita`: Basi Molecolari dell’ipotiroidismo congenito:

predizione, prevenzione ed intervento I.S.S Centro Eccellenza

Ambi-SEN, Pisa.

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