Sorting nexins (SNXs) are a large group of proteins defined by presence of a conserved phox homology (PX) domain, which mediates targeting to plasma membrane or endomembrane system via association with phosphatidylinositol phospholipids [1; 2]. SNXs were reported exhibiting diverse functions in cellular protein trafficking and signaling regulation, as a result of containing diverse additional domains, including membrane tubulating bin/amphiphysin/rvs (BAR) domain for membrane remodeling, band4.1/ezrin/radixin/moesin (FERM) domain and postsynaptic density 95/discs large/zonula occludens-1 (PDZ) domain for cargo recognition, and regulators of G protein signaling homology (RH) domain for G protein signaling regulation [2; 3; 4; 5].

SNX13, SNX14 and SNX25 are a subgroup of sorting nexins (SNX-RH) that contain RH domain [5], which may involve in cellular signaling transduction [4]. Canonical RH domains fold as two helical bundles composed of nine helices, which serve as GTPase accelerating protein for Gα subunits by stabilizing the flexible switch regions, further leading to GPCR signaling termination [6; 7]. RGS proteins of R4, R7, R10 and RZ subfamilies are broadly reported canonical RH domains that play important roles in G protein signaling [7; 8; 9]. Besides canonical RH domains, non-canonical RH domains were also identified to not possess GAP activity. AXIN, an important scaffold protein in β-Catenin signaling, can directly interact with activated Gαs subunit and adenomatous polyposis coli (APC) tumor suppressor protein via its RH domain, but lacking of GAP activity [10; 11; 12]. RH domain of G protein-coupled receptor kinase GRK2 was also reported lacking of GAP activity, although it possessed binding capacity toward activated Gαq subunit, due to an unconventional binding mode with Gαq [7; 12; 13].

Among SNXs, SNX13, also named RGS-PX1, was the first protein identified to contain RH domain and plays bifunctional roles in regulating Gαs mediated signaling and promoting EGFR degradation, and therefore is essential for embryonic development [14; 15; 16]. Subsequently, SNX14 was determined as a negative regulator for signaling transduction of Gαs coupled receptor 5-HT6R [17], and was important for normal neuronal excitability and synaptic transmission [18]. It was also reported that loss-of-function mutation in SNX14 could cause cerebellar atrophy together with ataxia [19; 20]. Although the roles in G protein mediated signaling remain unknown, SNX25 was identified as a regulator in several cellular signaling pathways. In 2011, SNX25 was identified as a negative regulator for transforming growth factor β (TGF-β) signaling by promoting trafficking and degradation of this receptor [21]. TGF-β signaling pathway was proposed contributing to the epileptogenesis, and expression of SNX25 was increased in brain tissue of epilepsy patients and animal model with epilepsy of chronic phase, indicating SNX25 might correlate to the development of epilepsy [22]. More recently, SNX25 was also demonstrated as a negative regulator in NF-κB signaling pathway that prevents NF-κB translocation to nucleus by suppressing ubiquitination of IκBα [23].

Although several studies have addressed the roles of SNX-RH in signaling regulation, the mechanism details of this regulation are still unclear. To better understand the precise function of SNX-RH in cellular signaling transduction, here, we focused on structural studies on RH domains of SNX13, SNX14 and SNX25. We successfully determined crystal structures of SNX13-RH domain and SNX25-RH domain, both exhibiting as non-canonical RH domains that is consistent with previously proposed by Tesmer [5]. Crystal structures reveal a homodimer of SNX13-RH mediated by extended α4 and α5 helices, and a thiol modulated homodimer of SNX25-RH triggered by a C362 residue on α6 helix. Structural and biochemical studies further demonstrated that RH domains of SNX-RH, lacking the critical residues for interaction, do not bind with Gα subunits. Thus, our results suggest that RH domains of SNX-RH cannot regulate GPCR signaling via GAP activity but may function as a dimerization module for SNX-RH.