# A single domain intrabody specifically targeting the follicle-stimulating hormone receptor (FSHR) affects receptor signaling and trafficking

Pauline Raynaud, Juliette Gourdon, Frédéric Jean-Alphonse, Lucille Berthet, Camille Gauthier, Vinesh Jugnarain, Océane Vaugrente, Thomas Boulo, Amandine Vallet, Christophe Gauthier, et al.

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## To cite this version:

Pauline Raynaud, Juliette Gourdon, Frédéric Jean-Alphonse, Lucille Berthet, Camille Gauthier, et al. A single domain intrabody specifically targeting the follicle-stimulating hormone receptor (FSHR) affects receptor signaling and trafficking. 4th IRN i-GPCRnet meeting, Jul 2024, Nottingham, United Kingdom. **hal-04731960**

HAL Id: hal-04731960

https://hal.inrae.fr/hal-04731960v1

Submitted on 11 Oct 2024

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# A single domain intrabody specifically targeting the follicle-stimulating hormone receptor (FSHR) affects receptor signaling and trafficking

P. Raynaud*¹, J. Gourdon¹, F. Jean-Alphonse¹⁻², Lucille Berthet¹, Ca. Gauthier¹, V. Jugnarain¹, O. Vaugrente¹, T. Boulo¹, A. Vallet¹, Ch. Gauthier¹, D.

Klett¹, Y. Combarnous¹, A. Hanyaloglu³, E. Reiter¹⁻², G. Bruneau¹, P. Crépieux¹⁻²

¹INRAE, Nouzilly, France   ²Inria, Palaiseau, France   ³Imperial College London, London, United Kingdom

## INTRODUCTION

The follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor (GPCR) involved in the growth and maturation of ovarian follicles and in spermatogenesis. Therefore, it is the main target of medically-assisted therapies, and also a putative target for hormonal contraception.

At a cellular level, FSH-activated FSHR primarily activates the Gs protein pathway. Following GRK-phosphorylation, FSHR then recruits β-arrestins, leading to its internalization to the early endosome (EE), but mainly to the very EE, from which it is quickly recycled back to the plasma membrane.

Intracellular variable fragments from heavy-chain antibody from camelids (intra-VHH) provide a better understanding on the relationships between receptor conformation and signaling activity, but so far, this property has been explored only for five GPCRs (Raynaud et al., 2022).

Intra-VHHs targeting the FSHR with different functional effects constitute precious tools to correlate discrete conformations of the FSHR with the signaling network engaged and the physiological outcome.

<figure><img src="image_3.png" /></figure>

## INTRA-VHH SELECTION

<figure><img src="image_4.png" /></figure>

Intra-VHH selection was performed by phage display. FSHR-expressing HEK293 cell membrane fragments were used, in presence or absence of FSH, with the intracellular side oriented to the phage library.

The VHHs carried by the phages recognising the antigens were sequenced by NGS, and sequences were aligned, clustered and compared to control conditions in order to identify specific clones.

This strategy allowed the selection of an intra-VHH of interest: iPRC2

## INTRA-VHH CHARACTERIZATION

### 1 Binding properties

<figure><img src="image_5.png" /><figcaption>**A. Binding by BRET**</figcaption></figure>

HEK293
iPRC2

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Stimulation with 3,3 nM of FSH, 3,3 nM of LH, 100 nM AVP,

<figure><img src="image_8.png" /></figure>

→ iPRC2 specifically binds the FSHR in presence of FSH or not

<figure><img src="image_9.png" /><figcaption>**B. Epitope mapping by HTRF**</figcaption></figure>

<figure><img src="image_10.png" /></figure>

→ iPRC2 recognizes FSHR ICL1 and ICL3

### 3 FSHR trafficking

<figure><img src="image_11.png" /><figcaption>**A. β-arrestins**</figcaption></figure>

→ iPRC2 has no effect on β-arrestin 2 recruitment

<figure><img src="image_12.png" /><figcaption>**C. FSHR Recycling**</figcaption></figure>

→ iPRC2 decreases FSHR recycling

<figure><img src="image_13.png" /><figcaption>**B. FSHR intracellular trafficking**</figcaption></figure>

<figure><img src="image_14.png" /></figure>

→ iPRC2 does not impact FSHR internalisation but increases its trafficking to the early endosomes (EE)

=> Location biais

### 2 G protein signaling pathway (ICL3 being involved in Gs protein recruitment)

<figure><img src="image_15.png" /><figcaption>**A. cAMP production**</figcaption></figure>

<figure><img src="image_16.png" /></figure>

→ iPRC2 decreases G protein-dependent cAMP production in response to FSH, but have no effect on ADRB2 response to isoproterenol (ISO)
→ It reduces CRE-dependent transcription in response to FSH

<figure><img src="image_17.png" /><figcaption>**B. CRE-dependent transcription**</figcaption></figure>

### 4 Role of APPL1

The protein APPL1 interacts with FSHR on ICL1, and is involved in FSHR recycling when phosphorylated by the PKA.

What is the phosphorylation status of APPL1 ?

→ iPRC2 induces a decrease of APPL1 phosphorylation

<figure><img src="image_18.png" /><figcaption>**Mechanistic hypotheses regarding the recycling defect of FSHR**</figcaption></figure>

<figure><img src="image_19.png" /></figure>

① iPRC2 impairs APPL1 phosphorylation as a result of the reduced cAMP

② iPRC2 could also directly interfere with APPL1 binding to the FSHR

## CONCLUSIONS AND PERSPECTIVES

In summary, iPRC2

* reduces cAMP production (competition ?)

* increases its trafficking to the EE

* and induces a default in FSHR recycling

* potentially because it impairs APPL1 phosphorylation

Several questions still need to be addressed :

* Do FSHR and iPRC2 colocalize in the EE ?

* Is FSHR trafficking to the VEE decreased ?

* Is the cAMP production reduced by a competition of iPRC2 with the Gs protein ?

* increases its trafficking to the EE

* Is FSHR interaction with APPL1 impaired by iPRC2 ?

<figure><img src="image_20.png" /></figure>