Structural and Functional Insights into the Regulation of P-Rex1, a Key Regulator of Neutrophil Chemotaxis and Cancer Metastasis

Kernkonzepte

P-Rex1, a guanine nucleotide exchange factor (GEF) for Rac, is synergistically activated by PIP3 and Gβγ subunits, but its regulation remains poorly understood. The study reveals that IP4 inhibits P-Rex1 activity by inducing a conformational change where the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain, stabilized by interactions between the DEP1 and DH domains, and between the PH domain and a 4-helix bundle subdomain.

Zusammenfassung

The study investigates the regulation of P-Rex1, a key guanine nucleotide exchange factor (GEF) for Rac that plays central roles in neutrophil chemotaxis and cancer metastasis. P-Rex1 is synergistically activated by PIP3 and the heterotrimeric Gβγ subunits, but the mechanistic details of its regulation remain poorly understood.

The researchers discovered that Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP4 complex revealed a conformation where the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain, and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1.

Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells.

Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. The results suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

Statistiken

Ins(1,3,4,5)P4 (IP4) induces large decreases in backbone dynamics in diverse regions of the P-Rex1 protein.

Zitate

"Cryo–electron microscopy analysis of the P–Rex1·IP4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain."
"Interactions with PIP3–containing liposomes led to disruption of these interfaces and increased dynamics protein–wide."

Wichtige Erkenntnisse aus

Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4

by Ravala,S. K.... um www.biorxiv.org 09-16-2023

https://www.biorxiv.org/content/10.1101/2023.09.15.557836v3

Tiefere Fragen

How do the structural changes in P-Rex1 induced by PIP3 and IP4 binding affect its interactions with other regulatory proteins or signaling pathways?

The structural changes in P-Rex1 induced by PIP3 and IP4 binding play a crucial role in modulating its interactions with other regulatory proteins and signaling pathways. Upon binding of PIP3, P-Rex1 undergoes conformational changes that disrupt the autoinhibited state, leading to increased dynamics protein-wide. This activation allows P-Rex1 to interact with the heterotrimeric Gβγ subunits, further enhancing its GEF activity for Rac. On the other hand, IP4 binding inhibits P-Rex1 activity by inducing large decreases in backbone dynamics and stabilizing a conformation where the PH domain occludes the DH domain's active site. This inhibited state prevents P-Rex1 from interacting with downstream effectors and inhibits its GEF activity. Therefore, the structural changes induced by PIP3 and IP4 binding directly impact P-Rex1's ability to interact with other regulatory proteins and signaling pathways, ultimately influencing cellular processes such as chemotaxis and cancer metastasis.

What are the potential therapeutic implications of targeting the P-Rex1 regulatory mechanisms for the treatment of neutrophil-mediated diseases or cancer metastasis?

Targeting the regulatory mechanisms of P-Rex1 presents promising therapeutic implications for the treatment of neutrophil-mediated diseases and cancer metastasis. Since P-Rex1 plays a central role in regulating Rac activity and cell migration, inhibiting its GEF activity could potentially impede neutrophil chemotaxis, which is crucial in inflammatory responses. In the context of cancer metastasis, targeting P-Rex1 could hinder the migration and invasion of cancer cells, thereby reducing metastatic spread. By understanding the structural changes that regulate P-Rex1 activity, novel therapeutic strategies could be developed to modulate its function selectively. Small molecules or peptides that disrupt the autoinhibited state of P-Rex1 or stabilize its inhibited conformation could serve as potential therapeutic agents for treating diseases involving aberrant P-Rex1 activity.

Could the insights into P-Rex1 regulation be applied to understand the regulation of other GEF proteins involved in cell migration and cancer progression?

The insights gained from studying P-Rex1 regulation could indeed be applied to understand the regulation of other GEF proteins involved in cell migration and cancer progression. GEF proteins play critical roles in activating small GTPases like Rac, which are key regulators of cell migration and invasion in cancer. The mechanisms by which P-Rex1 is activated by PIP3 and inhibited by IP4, leading to conformational changes that modulate its activity, may be shared by other GEF proteins. Understanding how structural changes regulate GEF activity can provide valuable insights into the broader regulatory mechanisms of GEF proteins in cell migration and cancer progression. By extrapolating the findings from P-Rex1 to other GEFs, researchers can uncover common themes in GEF regulation and potentially identify new targets for therapeutic intervention in diseases characterized by dysregulated cell migration and metastasis.