Description

In vertebrates, the Attractin (Atrn) transmembrane protein family is comprised of ATRN, Attractin-like 1 (ATRNL1) and Multiple epidermal growth factor-like domain 8 (MEGF8) proteins, which contain an extensive extracellular domain with numerous protein interaction motifs and a highly conserved intracellular MASRPF motif (Fig. 1A and B) (Walker et al., 2007). Common to all three paralogs is that they have a strong phenotypic overlap with E3-ligase Mahogunin ring finger 1 (Mgrn1), due to regulation of the same G-protein coupled receptors (GPCRs) as Mgrn1. For example, rodent Atrn and Mgrn1 negatively regulate signaling melanocortin receptors MCR1 and MCR4 by promoting their endolysosomal trafficking. Thus, loss of function mutations in Atrn or Mgrn1 suppress the pigmentation and obesity phenotypes of MCR1/4 antagonist gain of function mutants (Barsh et al., 2002; He et al., 2003; Nagle et al., 1999). Loss of function of Megf8 and Mgrn1 mutants exhibit common developmental phenotypes (Aune et al., 2008; Cota et al., 2006; Zhang et al., 2009) and both proteins were identified as negative regulators of the GPCR Smoothened in mice (Pusapati et al., 2018). Most recently it was shown in vitro that Mgrn1 and Megf8 co-immunoprecipitate in a MASRPF motif-dependent manner, and this association is required for Mgrn1 to mediate membrane-tethered ubiquitination to modulate the signaling strength of Hedgehog morphogens in mice (Kong et al., 2020).

Distracted (dsd) is the sole Drosophila ortholog of human ATRN and ATRNL1 (Fig. 1A), while Drosophila Megf8, like rodent Megf8, is required for early development (Lloyd et al., 2018). In contrast to Drosophila Megf8, the MASRPF motif is fully conserved in Dsd (Fig. 1B). The DRSC Integrative Ortholog Prediction Tool uses 15 different algorithms to identify orthologous genes (https://www.flyrnai.org/cgi-bin/DRSC_orthologs.pl, Version 8.0, August 2019). For CG9941 (hereafter referred to as dMrgn1) more algorithms predicted that it is orthologous to mammal MGRN1 (human 11/15, rat 7/13, mouse 10/15) than for RNF157 (Ring Finger Protein 157; human 10/14, rat 4/15, mouse 10/15). While the zinc finger domain is highly conserved (83% amino acid identity) in dMgrn1 when compared to either mammal MGRN1 or RNF157 proteins, the entire protein is less well preserved (amino acid identity 35-37%/similarity 48-51%) in part due to the Drosophila protein being larger by approximately 210 amino acids when compared to mammalian Mgrn1.

Here, we generated myc-tagged Dsd containing (UAS-Dsd-myc) or lacking the MASRPF motif (UAS-DsdΔMASR-myc, Fig. 1B), and FLAG-tagged dMgrn1 (UAS-dMgrn1-FLAG) transgenic lines to determine whether Dsd and dMgrn1 are in a complex together in vivo, and if the interaction is dependent on the MASRPF motif. We panneuronally (nSyb-Gal4) expressed dMgrn1-FLAG alone or together with Dsd-myc or DsdΔMASR-myc, and used Drosophila heads to perform immunoprecipitation (IP) with myc-trap agarose beads. In western blots of fly head supernatant prior to IP, dMgrn1-FLAG is detected at 110 kDa and Dsd-myc at 160kDa (Fig. 1, lane 1-3) confirming protein expression. Multiple independent IP experiments were performed using two distinct transgenic lines for expression of Dsd-myc and DsdΔMASR-myc, while using the same transgenic line for expression of dMgrn1-FLAG. The results demonstrated that Dsd and dMgrn1 co-IP, suggesting they interact in vivo (n=4), and that this interaction was indeed dependent on the MASRPF motif (n=3, Fig. 1C). After co-IP, dMgrn1-FLAG protein was detected abundantly when it was co-expressed with Dsd-myc (Fig. 1C, lane 6) but absent or at similarly low levels as the negative controls (UAS-dMgrn1-FLAG,nSyb-Gal4, Fig. 1C, lane 4) when co-expressed with DsdΔMASR-myc (Fig. 1C, lane 5). These results imply that Drosophila is a valid model to study ATRN’s proposed role as a substrate adaptor for MGRN1 (Kong et al., 2020). Loss of function of Atrn and Mgrn1 in rodents is associated with mitochondrial dysfunction, elevated oxidative stress, and adult-onset spongiform neurodegeneration (Bronson et al., 2001; Cota et al., 2008; He et al., 2003; Kuramoto et al., 2001; Li et al., 2014; Muto and Sato, 2003; Paz et al., 2007). In humans, ATRN is a candidate gene for sporadic amyotrophic lateral sclerosis (Morahan et al., 2009), ATRN plasma levels are altered in early-onset Alzheimer’s disease (AD) patients prior to symptoms (Muenchhoff et al., 2016), and its gene expression is reduced in Parkinson disease (PD) patients (Glaab and Schneider, 2015). Since the ATRN/MGRN1 receptor substrates involved in nervous system survival have not been identified, future studies in Drosophila may provide insight into the relevant signaling pathways.