The Lipid-binding PX Domain of RRC-1 (ARHGAP32/33) is Required for Optimal Assembly and Function of Integrin Adhesion Complexes at the Muscle Cell Boundary in C. elegans

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Abstract Integrin adhesion complexes (IACs) are a network of many proteins that serve as anchors of the cell to the extracellular matrix (ECM). In muscle, IACs located at costameres, also serve to transmit the force of muscle contraction to the outside of the cell. We have reported that IACs, which are found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the RacGEF PIX-1 for their proper assembly or maintenance. We have reported that a RacGAP for the PIX pathway is RRC-1, is in a complex with PIX-1, and that RRC-1 is required for assembly or maintenance of IACs at MCBs. Our previous studies suggested that RRC-1 might be associated with the muscle cell membrane, and here we present evidence that this occurs via its PX domain, a domain that is known to bind to membrane phosphoinositides (PIPs). We predict the existence of a PX domain based on bioinformatic analysis and AlphaFold3, which includes conserved residues characteristic of most PX domains and a PIP binding site. This region of RRC-1 binds to phosphoinositides in vitro. Analysis of a nematode strain that has an in-frame deletion of the PX domain, indicates that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffold protein GIT-1. Lastly, we show that the overexpression of the full length RRC-1, but not RRC-1 with an in-frame deletion of its PX domain, results in reduced accumulation of IAC components and reduced whole animal movement. Our study highlights the importance of RRC-1’s lipid interactions at the cell membrane for proper assembly and function of IACs in C. elegans muscle. Article Summary Integrin adhesion complexes (IACs) facilitate the transmission of force of muscle contraction to the outside of the cell. IACs, found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the PIX-1 (a RacGEF) signaling pathway for their assembly or maintenance. A RacGAP for the PIX pathway is RRC-1. Here, we show that RRC-1 has a PX domain that binds to membrane phosphoinositides. We also show that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffolding protein GIT-1.

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