Comparative Phosphoproteomic Profiling of Type III Adenylyl Cyclase Knockout and Control, Male, and Female Mice.

Yuxin Zhou,Ashley Sterpka,Feixia Chu,Xuanmao Chen,Haiying Wang,Liyan Qiu

doi:10.3389/fncel.2019.00034

Yuxin Zhou, Ashley Sterpka + Show 4 more

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https://doi.org/10.3389/fncel.2019.00034

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Abstract

Type III adenylyl cyclase (AC3, ADCY3) is predominantly enriched in neuronal primary cilia throughout the central nervous system (CNS). Genome-wide association studies in humans have associated ADCY3 with major depressive disorder and autistic spectrum disorder, both of which exhibit sexual dimorphism. To date, it is unclear how AC3 affects protein phosphorylation and signal networks in central neurons, and what causes the sexual dimorphism of autism. We employed a mass spectrometry (MS)-based phosphoproteomic approach to quantitatively profile differences in phosphorylation between inducible AC3 knockout (KO) and wild type (WT), male and female mice. In total, we identified 4,655 phosphopeptides from 1,756 proteins, among which 565 phosphopeptides from 322 proteins were repetitively detected in all samples. Over 46% phosphopeptides were identified in at least three out of eight biological replicas. Comparison of AC3 KO and WT datasets revealed that phosphopeptides with motifs matching proline-directed kinases' recognition sites had a lower abundance in the KO dataset than in WTs. We detected 14 phosphopeptides restricted to WT dataset (i.e., Rabl6, Spast and Ppp1r14a) and 35 exclusively in KOs (i.e., Sptan1, Arhgap20, Arhgap44, and Pde1b). Moreover, 95 phosphopeptides (out of 90 proteins) were identified only in female dataset and 26 only in males. Label-free MS spectrum quantification using Skyline further identified phosphopeptides that had higher abundance in each sample group. In total, 204 proteins had sex-biased phosphorylation and 167 of them had increased expression in females relative to males. Interestingly, among the 204 gender-biased phosphoproteins, 31% were found to be associated with autism, including Dlg1, Dlgap2, Syn1, Syngap1, Ctnna1, Ctnnd1, Ctnnd2, Pkp4, and Arvcf. Therefore, this study also provides the first phosphoproteomics evidence suggesting that gender-biased post-translational phosphorylation may be implicated in the sexual dimorphism of autism.

Highlights

Primary cilia are tiny microtubule-based, membrane-ensheathed signaling devices present in most mammalian cells (Singla and Reiter, 2006)
Because AC3 is associated with major depressive disorder (MDD) (Wray et al, 2012; Chen et al, 2016) and autism spectrum disorders (ASD) (Skafidas et al, 2014; Yuen et al, 2017), we chose the prefrontal cortex, a brain region important for social behaviors, personality and emotion, and mood state (Siddiqui et al, 2008), in our phosphoproteomics analysis
These results are consistent with previous reports (Kasahara et al, 2014; Sipos et al, 2018; Sterpka and Chen, 2018), showing that AC3 is primarily confined to neuronal primary cilia in the brain

Summary

Introduction

Primary cilia are tiny microtubule-based, membrane-ensheathed signaling devices present in most mammalian cells (Singla and Reiter, 2006). Neuronal primary cilia have abundant expression of G-protein coupled receptors (GPCRs), such as type 3 somatostatin receptor (Wang et al, 2009; Einstein et al, 2010), type 6 serotonin receptor (Brodsky et al, 2017), and melaninconcentrating hormone receptor 1 (Green et al, 2012). This suggests that neuronal primary cilia depend on metabotropic signal pathways, rather than electrical input from synapses, to modulate neuronal activity. If not all, ciliary GPCRs (Schou et al, 2015) are found to be either Gαs- or Gαi-protein coupled receptors (Qiu et al, 2016; Sterpka and Chen, 2018), which rely on heterotrimeric G-proteins, adenylyl cyclases, and cyclic adenosine monophosphate (cAMP) to send signals to the soma of neurons (Qiu et al, 2016; Sterpka and Chen, 2018)

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