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Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains

Citation

Nano, Patricia R. et al. (2021), Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains, Dryad, Dataset, https://doi.org/10.5061/dryad.dz08kprv9

Abstract

ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.

Methods

Data was generated from NIH-3T3 fibroblasts expressing a Gli-dependent green-fluorescent reporter that were retrovirally transduced with a library of mCherry-tagged ARHGAP36 isoform 2 variants. Cells expressing an ARHGAP36 mutant were isolated using a fluorescence-activated cell sort (FACS) based on mCherry fluorescence. A population of ARHGAP36-expressing cells were maintained as a “pre-selection” population, while the remaining cells were cultured in Hedgehog signaling-competent conditions. Cells harboring ARHGAP36 mutants that could not activate Gli proteins were isolated using FACS for mCherry+/EGFP– cells (post-selection population). Inactivating point mutations were identified by amplifying ARHGAP36 inserts from the genomic DNA of isolated cell populations and sequencing the resulting 1.6-kb amplicons on an Illumina NextSeq 500 Sequencer using High-Output v2 kits to produce paired-end, 150-bp reads. FASTQ files were processed by first using Bowtie v2.3 to align reads to an index built from the wild-type ARHGAP36 coding sequence (MtInsert_2_Amplicon_ARHGAP36.fasta). SAMtools v1.3.1 was used to sort the mapped reads by name and convert to a SAM file format. A custom Python script was used to calculate nucleotide and amino acid mutation frequencies.