Tài liệu Báo cáo khoa học: Role of cleavage and shedding in human thyrotropin receptor function and

Role of cleavage and shedding in human thyrotropin receptor function

and trafficking

Myle` ne Quellari1

, Agne` s Desroches1

, Isabelle Beau1

, Emmanuelle Beaudeux1 and Micheline Misrahi1,2

1

INSERM E120, Re´cepteurs, Signalisations et Physiopathologie Thyroı¨dienne et de la Reproduction, and 2

Laboratoire

d’Hormonologie et Biologie Mole´culaire, Hoˆpital Biceˆtre, IFR Biceˆtre, Le Kremlin Biceˆtre, France

The thyrotropin receptor (TSHR) undergoes a cleavage at

the cell membrane, leading to a heterodimer, comprising an

a extracellular and a b-transmembrane and intracellular

subunits, held together by disulfide bonds.Moreover, part of

the a-subunit of the receptor is shed from thyroid and

transfected L cells.To understand the role of cleavage and

shedding, we constructed deletion mutants starting,

respectively, at the most N-terminal (S314), and C-terminal

(L378) cleavage sites previously mapped, corresponding to

free b1 or b2-subunits without further modification of

receptor structure.Functional studies performed in COS-7

cells showed that both mutants display an increased basal

activation of the cAMP pathway when compared with the

wild-type receptor.By contrast, deletion of almost the entire

extracellular domain of the receptor (TM409 mutant) totally

impairs receptor function, thus confirming a role of the

juxtamembrane extracellular region in receptor function.

The b1 mutant receptor exhibited an increased internalizat￾ion when compared with the hormone-activated holo￾receptor.Furthermore, no recycling was observed in the case

of the b1 mutant receptor.These observations strongly argue

for a different conformation between the receptor activated

by cleavage and shedding on the one hand, and the receptor

activated by the ligand on the other hand.Cleavage and

shedding of a receptor already activated by a transmem￾brane activating mutation M453T further increase its

activity, showing that the extracellular domain still exerts a

negative effect in the M453T holoreceptor.An increased

internalization of the M453T receptor was observed when

compared with the wild-type receptor, which was increased

further in the corresponding truncated b1-M453T receptor.

Thus cleavage and shedding yield TSHR activation but also

increase internalization of the free b-subunits of the receptor,

the latter mechanism limiting simultaneously excessive

receptor signaling.The combined effects may be responsible

for the limited basal constitutive activation of the cAMP

pathway that is detected for the TSHR.

Keywords: thyrotropin receptor; cleavage; shedding; con￾stitutive activity; traffic.

The thyrotropin receptor (TSHR) plays a key role in

thyroid growth and function [1–3].This receptor is also the

target of stimulating or blocking autoantibodies in patients

with autoimmune diseases [4,5].The TSHR belongs to a

particular subgroup of G protein-coupled receptors, inclu￾ding the FSH and LH receptors [6].They are characterized

by the presence of a seven transmembrane domain and a

large extracellular domain involved in high affinity hormone

binding.These three receptors are mainly coupled to Gs,

leading to the activation of the adenylate cyclase pathway.

However, unlike the gonadotropin receptors, the TSHR

transduces a signal via adenylate cyclase even in the absence

of ligand, thus having a weak constitutive activity [7].

The TSHR undergoes a unique post-translational mat￾uration among G protein-coupled receptors.In human

thyroid membranes, intramolecular cleavage occurring at

the cell surface generates two subunits: an approxi￾mately 53 kDa a extracellular subunit, and a wide approxi￾mately 33–42 kDa b-transmembrane and intracellular

subunit (called A- and B-subunits, according to the

terminology of Rees-Smith et al. [4,8]), held together by

disulfide bridges [9].A similar maturation is also observed in

an L cell line stably transfected with the human TSHR

cDNA [10].In addition, the extracellular domain of the

receptor is shed from the cell surface of thyroid and

transfected L cells [11].The shedding is an enzymatically

catalyzed process, where the disulfide bonds reduction is

performed by cell-surface associated protein disulfide iso￾merase [12].Other studies suggested that cleavage was

required for the formation of TSHR dimers and higher

order complexes [13].PDI activity may also account for

disulfide bonding of TSHR oligomers.

Immunopurification and microsequencing of the b-sub￾units in thyroid membranes and transfected L cells led to the

identification of the cleavage sites of the TSHR.In fact,

multiple cleavage sites exist [14,15].They are unrelated and

located in a specific extracellular region of the receptor (E3)

that displays no homology with the LH and FSH receptors

[15–17].In transfected L cells and in thyroid membranes, the

Correspondence to M.Misrahi, Inserm E120, Baˆtiment Gregory

Pincus, Hoˆpital Biceˆtre, 94275, Le Kremlin Biceˆtre, France.

Fax: + 33 1 45213822, Tel.: + 33 1 45212746/49591828,

E-mail: [email protected]

Abbreviations: ABTS, 2,2¢-azine-di(ethylbenzthiazoline sulfonate);

bTSH, bovine TSH; FSH, follicle stimulating hormone; GPCR,

G protein-coupled receptor; LH, luteinizing hormone; TSH,

thyrotropin hormone; TSHR, thyroid stimulating hormone receptor.

(Received 25 March 2003, revised 7 June 2003,

accepted 12 June 2003)

Eur. J. Biochem. 270, 3486–3497 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03718.x