NHERF2/SIP-1 Interacts with Mouse SRY via a Different Mechanism than Human SRY

Laurie Thevenet,Kenneth H Albrecht,Safia Malki,Philippe Berta,Brigitte Boizet-Bonhoure,Francis Poulat

doi:10.1074/jbc.m504127200

Abstract

In mammals, male sex determination is controlled by the SRY protein, which drives differentiation of the bipotential embryonic gonads into testes by activating the Sertoli cell differentiation program. The morphological effects of SRY are well documented; however, its molecular mechanism of action remains unknown. Moreover, SRY proteins display high sequence variability among mammalian species, which makes protein motifs difficult to delineate. We previously isolated SIP-1/NHERF2 as a human SRY-interacting protein. SIP-1/NHERF2, a PDZ protein, interacts with the C-terminal extremity of the human SRY protein. Here we showed that the interaction of SIP-1/NHERF2 and SRY via the SIP-1/NHERF2 PDZ1 domain is conserved in mice. However, the interaction occurs via a domain that is internal to the mouse SRY protein and involves a different recognition mechanism than human SRY. Furthermore, we show that mouse and human SRY induce nuclear accumulation of the SIP-1/NHERF2 protein in cultured cells. Finally, a transgenic mouse line expressing green fluorescent protein under the control of the mouse Sry promoter allowed us to show that SRY and SIP-1/NHERF2 are co-expressed in the nucleus of pre-Sertoli cells during testis determination. Taken together, our results suggested that the function of SIP-1/NHERF2 as an SRY cofactor during testis determination is conserved between human and mouse.

Highlights

In mammals, testicular differentiation is under the control of the Y chromosome-encoded master switch gene SRY (1–3), which instructs the supporting cell precursors to become Sertoli cells rather than granulosa cells (4)
Despite the strong homology shared by the SIP-1 PDZ1 and PDZ2 domains, an interaction was not detected between mouse SRY (mSRY) and GST-PDZ2 (Fig. 1B, lane 3), suggesting an important selectivity of interaction
In our original study using the yeast two-hybrid interaction test, Human SRY (hSRY) was interacting with PDZ2, in contrast with our results presented here

Summary

Introduction

Testicular differentiation is under the control of the Y chromosome-encoded master switch gene SRY (1–3), which instructs the supporting cell precursors to become Sertoli cells rather than granulosa cells (4). The mammalian SRY proteins can be subdivided into two groups, one including the non-rodent mammals where the HMG domain is central, and the second including the rodent species, where the N-terminal domain is restricted to two amino acids directly followed by the HMG domain and a long C-terminal domain made of at least two different regions. Recent transgenic experiments, where expression of the human SRY protein was controlled by mouse Sry regulatory regions, show the capacity of the human protein to induce testis determination in a mouse context (18) These results suggest two possible hypotheses in regard to the functionality of the different SRY protein domains. Rapid evolution of SRY genes across mammalian species argues in favor of this hypothesis, but sex-reversing mutations outside the HMG box in humans argue against it

Methods

Results

Conclusion