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ArreSTick Motif is Responsible for GPCR-B-Arrestin Binding Stability and Extends Phosphorylation-Dependent B-arrestin Interactions to Non-Receptor Proteins

ABSTRACT: The binding and function of B-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. To investigate this pattern, we employed a 1D sequence convolution model trained on a dataset of GPCRs that have established B-arrestin binding properties. This approach allowed us to identify the amino acid pattern required for GPCRs to form stable interactions with B-arrestins. This motif was named 'arreSTick.' Our data show that the model predicts the strength of the coupling between GPCRs and B-arrestins with high accuracy, as well as the specific location of the interaction within the receptor sequence. Furthermore, we show that the arreSTick pattern is not limited to GPCRs, and is also present in numerous non-receptor proteins. Using a proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between numerous non-receptor proteins and B-arrestins. For example, the HIV-1 Tat Specific Factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and our data show that its subcellular localization is influenced by its coupling to B-arrestin2. Our findings unveil a broader regulatory role for B-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.

INSTRUMENT(S): maXis II

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Gabor Turu

PROVIDER: MSV000092868 | MassIVE |

SECONDARY ACCESSION(S): PXD045420

REPOSITORIES: MassIVE

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Project description:Previous studies of E2F family members have suggested that protein-protein interactions may be the mechanism by which E2Fs are recruited to specific genomic regions. We have addressed this hypothesis on a genome-wide scale using ChIP-seq analysis of MCF7 cell lines that express tagged wildtype and mutant E2F1 proteins. First, we performed ChIP-seq for tagged wt E2F1. Then, we analyzed E2F1 proteins that lacked the N terminal SP1 and cyclin A binding domains, the C terminal transactivation and pocket protein binding domains, and the internal marked box domain. Surprisingly, we found that the ChIP-seq patterns of the mutant proteins were identical to that of wt E2F1. However, mutation of the DNA binding domain abrogated all E2F1 binding to the genome. These results suggested that the interaction between the E2F1 DNA binding domain and a consensus motif may be the primary determinant of E2F1 recruitment. To address this possibility, we analyzed the in vivo binding sites for the in vitro-derived consensus E2F1 motif (TTTSSCGC) and also performed de novo motif analysis. We found that only 12% of the ChIP-seq peaks contained the TTTSSCGC motif. De novo motif analysis indicated that most of the in vivo sites lacked the 5M-CM-"M-BM-^@M-BM-^Y half of the in vitro derived consensus, having instead the in vivo consensus of CGCGC. In summary, our findings do not provide support for the model that protein-protein interactions are involved in recruiting E2F1 to the genome, but rather suggest that recognition of a motif found at most human promoters is the critical determinant. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf 9 total ChIP-seq datasets; four different HA-ER-E2F1 mutants, and one HA ER E2F1 wild type dataset done in duplicate, from 5 different stable cell lines derived from MCF7 cells; Three Input replicates from 2 different stable cell lines derived from MCF7 cells; HA ER E2F1 wild type duplicate dataset from MCF7 stable cells; 1 HA ER E2F1 DBDmut replicate from MCF7 stable cells cells, 1 HA ER E2F1M-CM-^NM-BM-^TC replicate from MCF7 stable cells cells, 1 HA ER E2F1M-CM-^NM-BM-^TN/C replicate from MCF7 stable cells cells, 1 HA ER E2F1M-CM-^NM-BM-^TMB replicate from MCF7 stable cells cells, 1 HA ER E2F1M-CM-^NM-BM-^TMB replicate from MCF7 stable cells cells, 3 Input replicates from MCF7 stable cells cells.

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ArreSTick Motif is Responsible for GPCR-B-Arrestin Binding Stability and Extends Phosphorylation-Dependent B-arrestin Interactions to Non-Receptor Proteins

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