Gonzalez Ramirez, M. | Search bioRxiv

bioRxiv2025

Activated protein C promotes beta-arrestin-2- and c-Src-dependent caveolin-1 (Cav1) phosphorylation and alters Cav1 association with PAR1 and GRK5

G protein-coupled receptors (GPCRs) display bias towards either G proteins or GPCR kinase (GRK)-mediated {beta}-arrestin signaling depending on the agonist stabilized receptor conformation and cellular context. The cellular location of GPCRs particularly within plasma membrane microdomains such as lipid rafts contributes to biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits biased signaling in response to thrombin and activated protein C (APC). APC-induced {beta}-arrestin-2 ({beta}arr2) biased signaling requires PAR1 compartmentalization in caveolae, a subtype of lipid rafts, whereas thrombin-activated PAR1 G protein signaling does not. Caveolin-1 (Cav1) is the principal structural protein of caveolae and regulates signaling through protein-protein interactions. The mechanisms by which Cav1 contributes to APC/PAR1-induced {beta}arr2 biased signaling is not known. Here we report that APC-activated PAR1 modulates Cav1 phosphorylation via a {beta}arr2- and c-Src-dependent pathway. APC also regulates the dynamics of endogenous PAR1-Cav1 and GRK5-Cav1 co-localization examined by single molecule super-resolution stochastic optical reconstruction microscopy imaging in human cultured endothelial cells. We further demonstrate that GRK5 interacts with Cav1 in intact cells through an N-terminus aromatic-rich consensus Cav1 binding motif. Unlike wildtype GRK5, a GRK5 mutant defective in Cav1 binding localized predominantly to the cytoplasm rather than the plasma membrane and failed to promote {beta}arr2 recruitment to APC-activated PAR1. These studies suggest that Cav1 itself contributes to the regulation of APC-activated PAR1 {beta}arr2 biased signaling likely through multiple mechanisms that may converge on GRK5.

cell biology↗

bioRxiv2025

Plasmodium falciparum exploits NUAK1 to establish infection in human erythrocytes

The malaria parasite Plasmodium falciparum continues to demonstrate growing drug resistance, raising the need for innovative treatments. Host-directed therapeutics are emerging as a promising approach for many infectious diseases, but knowledge of critical host factors for malaria is limited. P. falciparum is an obligate intracellular parasite of human erythrocytes, suggesting it has evolved to exploit specific host pathways to establish infection. Here, we report that the AMPK-related kinase NUAK1 is a critical host factor for P. falciparum in erythrocytes and has potential as a therapeutic target. We show that NUAK1 is present in human erythrocytes and undergoes increased phosphorylation in P. falciparum-infected cells. Two highly selective NUAK1 inhibitors, HTH-01-015 and WZ4003, inhibited P. falciparum growth throughout its asexual life cycle, including during erythrocyte invasion. Chemoproteomic profiling confirmed the inhibitors selectivity for human NUAK1. We further show that treatment with the inhibitors reduces phosphorylation of the well-characterized NUAK1 substrate MYPT1 in erythroid cells. Moreover, we find that genetic overexpression of NUAK1 in erythroid cells partially rescues both the signaling and invasion phenotypes elicited by the small molecule inhibitors. These results establish a critical role for the NUAK1 signaling pathway in P. falciparum-infected erythrocytes and highlight its potential as a vulnerable target for host-directed malaria control.

microbiology↗