Human follitropin receptor (FSHR) interacts with the adapter protein 14-3-3τ

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Abstract

The human follitropin (follicle stimulating hormone, FSH) receptor (FSHR) is a G protein-coupled receptor (GPCR). To identify cytoplasmic proteins that may regulate FSHR function, a yeast-based interaction trap was performed. A linked construct of the first and second intracellular loops (iL1-iL2 bait) of FSHR was used as bait and a human ovarian cDNA library was used as prey. Among the proteins identified that interacted with the bait was 14-3-3τ, a member of a family of homodimeric cytoplasmic adapter proteins. Human granulosa cells, the site of FSHR expression in the ovary, were found to contain 14-3-3τ. Importantly, 14-3-3τ co-immunoprecipitated with FSHR stably expressed in HEK 293 cells. Its association with FSHR was follitropin-dependent. Over-expression of 14-3-3τ resulted in a modest decrease of follitropin-induced cAMP accumulation. Collectively, these data support a role for 14-3-3τ in follitropin action. The finding that 14-3-3τ interacts with FSHR is novel and should lead to new insights into the regulation of GPCR in general and FSHR specifically.

Introduction

Follitropin acts on target cells in testis (Sertoli cells) and ovary (granulosa cells) via its cognate receptor to regulate reproduction (Dias et al., 2002). Follitropin receptor has seven transmembrane segments and is comprised of a large extracellular amine-terminal domain, three extracellular loops, three intracellular loops (iL1, iL2, iL3) and an intracellular carboxyl-terminal peptide (iL4). Following follitropin binding, adenylate cyclase is stimulated by the Gsα subunit of the heterotrimeric G protein complex. As a member of the GPCR family, occupied FSHR acts as a nucleotide exchange factor. As such, ligand binding effects a conformational change in the receptor (Schmidt et al., 2001), which induces the exchange of GTP for GDP from Gsα, which subsequently activates adenylate cyclase (Dias et al., 2002).

Although the canonical pathway of follitropin-mediated cellular activation is through the cAMP/protein kinase A (PKA) pathway, follitropin activates additional pathways normally regulated by growth factors. For example, follitropin treatment of rat granulosa cells stimulates the p38 mitogen activated protein kinase (MAPK) pathway (Maizels et al., 1998), and the extracellular signal-regulated kinase (ERK) pathway (Cameron et al., 1996). In the case of p38 MAPK, the mechanism appears to involve PKA. Additionally, at supraphysiological doses of follitropin, activation of protein kinase C (PKC) has also been seen (Quintana et al., 1994).

Gonadotropin-mediated Akt (protein kinase B, PKB) phosphorylation has been found to occur independently of the PKA pathway (Gonzalez-Robayna et al., 2000). Follitropin also stimulates the phosphorylation of FOXO1a (also known as FKHR), a downstream effector in the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway (Gonzalez-Robayna et al., 2000), (Cunningham et al., 2003), (Nechamen et al., in press). Moreover, cAMP activation of GTPases signal through downstream cascades including ERK 1/2, PI3K and p38 MAPK (Richards, 2001).

Proteins that interact with the intracellular domains of the human follitropin receptor are not well characterized. Although evidence of phosphorylation of the rat FSHR has been demonstrated (Hipkin et al., 1995), the kinases responsible have not been defined. Both iL1 and iL3 contain consensus sites for PKA and PKC (Ulloa-Aguirre and Timossi, 1998). It has been inferred that the G protein-coupled receptor kinases (GRK) are involved, based on other GPCR and indirectly from the actions of dominant negative kinase mutants (Lazari et al., 1999). However, direct interaction has not been demonstrated. Phosphorylation of the rat FSHR has been mapped in part to iL1 (Nakamura et al., 1998).

Additionally, iL2 has been implicated in Gs activation and in maintaining FSHR in an inactive state (Timossi et al., 2002). The 449ERW451 motif in iL2, analogous to the D/ERY motif in GPCR, is implicated in GPCR activation and G protein coupling. Mutation of this motif in gonadotropin releasing hormone receptor (Arora et al., 1997), α1b-adrenergic receptor (Scheer et al., 2000) and oxytocin receptor (Fanelli et al., 1999) had variable effects on signaling, depending on which amino acid (a.a.) was mutated and the a.a. substitution. Therefore, both loops appear to play functional roles in follitropin receptor physiology.

To identify FSHR intracellular domain interacting proteins, a yeast-based interaction trap was undertaken using a linked iL1-iL2 construct as bait. One of the proteins identified was the tau isoform of 14-3-3, establishing that gonadotropin receptors interact with the 14-3-3 adapter family.

The 14-3-3 proteins are 30 kDa homodimers which play a key role in signal transduction pathways, cell division and apoptosis (for review, see (Tzivion and Avruch, 2002), (van Hemert et al., 2001)). The 14-3-3 proteins modulate protein activity through a variety of mechanisms. Binding of 14-3-3 may prevent two proteins, such as BAD and Bcl2, from interacting (Tan et al., 2000). Alternatively, 14-3-3 may act as a scaffold to bring proteins together as in the case of Raf and PKC (van der Hoeven et al., 2000). Probably the most commonly identified mode of action of 14-3-3 proteins is to sequester proteins into inappropriate cell compartments, thereby inhibiting function. A corollary to this mechanism is that 14-3-3 frequently binds to phosphorylated proteins. Dephosphorylation of the protein results in release of 14-3-3, freeing the protein to be translocated or otherwise activated, an example of which is KSR1 and Raf-1 (Ory et al., 2003). In addition, 14-3-3 can directly regulate the catalytic activity of an enzyme (Sato et al., 2002).

The identification of 14-3-3τ in an interaction trap, as discussed in this report, establishes that gonadotropin receptors associate with the 14-3-3 adapter family.

Section snippets

Preparation of FSHR bait plasmids and screening in the yeast interaction trap

Growth and transformation of yeast for the interaction trap (also known as the two-hybrid screen) were performed as described in (Toby and Golemis, 2001). The yeast strain RFY231 (MATa, trp1, his3, ura3, leu2::6lexAop-LEU2) was transformed using the one step transformation method (Chen et al., 1992), with the β-galactosidase reporter plasmid pSH18-34 (kind gift of Dr. Steve Hanes, Wadsworth Center, Albany, NY) and the bait plasmid. The bait plasmid, iL1-iL2, was constructed with human FSHR

Interaction trap identifies 14-3-3τ as interacting partner with FSHR iL1-iL2

To identify interacting partners of either iL1 or iL2, an interaction trap screen was performed. Table 1 lists the proteins that were identified as interacting with the bait protein. One of the clones identified was the tau isoform of the 14-3-3 proteins. The full coding region of 14-3-3τ was not isolated in the cDNA insert. As seen in Fig. 2, a region corresponding to amino acids 50–246 was isolated. The absence of the N-terminal region, which is the dimerization region (van Hemert et al., 2001

Discussion

The identification of 14-3-3τ as an interacting partner was intriguing for several reasons. First, there is only a single serine residue in the second intracellular loop bait construct (S471), and this does not fall within a canonical 14-3-3 binding sequence (RSXpSXP) (van Hemert et al., 2001). Additionally, it is unclear if the bait protein would be phosphorylated in the context of the yeast interaction trap system. However, additional motifs are routinely being identified as 14-3-3 targets,

Acknowledgements

The authors gratefully acknowledge the expert technical assistance of Richard Thomas. Oligonucleotide synthesis and DNA sequencing were performed by the Molecular Genetics Core Facility. Hiroko Yoshinari, Linda O’Keefe and Gerald Kornatowski supplied the 293/FSHR cells. George Bousfield (Wichita State University, Wichita, KS) provided a human pituitary crude extract (GTN fraction) from which follitropin was purified in this laboratory. Research supported by NIH HD18407 and NIH F32HD08537 (BDC).

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