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Published on: July 9, 2014
Mutational analysis of the Aspergillus ambient pH receptor PalH underscores its potential as a target for antifungal
Daniel Lucena-Agell1, América Hervás-Aguilar1, Tatiana Múnera-Huertas2
1Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain.
Abstract:
The pal/RIM ambient pH signalling pathway is crucial for the ability of pathogenic fungi to infect hosts. The Aspergillus nidulans 7-TMD receptor PalH senses alkaline pH, subsequently facilitating ubiquitination of the arrestin PalF. Ubiquitinated PalF triggers downstream signalling events. The mechanism(s) by which PalH transduces the alkaline pH signal to PalF is poorly understood. We show that PalH is phosphorylated in a signal dependent manner, resembling mammalian GPCRs, although PalH phosphorylation, in contrast to mammalian GPCRs, is arrestin dependent. A genetic screen revealed that an ambient-exposed region comprising the extracellular loop connecting TM4-TM5 and ambient-proximal residues within TM5 is required for signalling. In contrast, substitution by alanines of four aromatic residues within TM6 and TM7 results in a weak 'constitutive' activation of the pathway. Our data support the hypothesis that PalH mechanistically resembles mammalian GPCRs that signal via arrestins, such that the relative positions of individual helices within the heptahelical bundle determines the Pro316-dependent transition between inactive and active PalH conformations, governed by an ambient-exposed region including critical Tyr259 that potentially represents an agonist binding site. These findings open the possibility of screening for agonist compounds stabilizing the inactive conformation of PalH, which might act as antifungal drugs against ascomycetes.
Insights
The fungal pathogen Aspergillus nidulans uses the PalH receptor to sense alkaline pH for host infection. This study reveals PalH
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The pal/RIM pathway regulates pathogenic fungal host infection.
- The 7-transmembrane domain (7-TMD) receptor PalH in Aspergillus nidulans senses alkaline pH.
- PalH signaling leads to arrestin PalF ubiquitination and downstream events, but the mechanism is unclear.
Purpose of the Study:
- To elucidate the signaling mechanism of the PalH receptor in response to ambient pH.
- To investigate the structural and functional relationship between PalH and arrestin PalF.
- To identify potential drug targets for antifungal therapies.
Main Methods:
- Phosphorylation analysis of PalH.
- Genetic screening to identify key regions of PalH involved in signaling.
- Site-directed mutagenesis to probe the function of specific amino acid residues.
Main Results:
- PalH phosphorylation is signal-dependent and arrestin-dependent.
- An extracellular loop (TM4-TM5) and TM5 residues are crucial for PalH signaling.
- Mutations in TM6 and TM7 aromatic residues lead to constitutive pathway activation.
- PalH conformation transitions are regulated by an ambient-exposed region including Tyr259.
Conclusions:
- PalH functions similarly to mammalian G protein-coupled receptors (GPCRs) but with unique arrestin-dependent phosphorylation.
- The relative orientation of transmembrane helices dictates PalH's active and inactive states.
- Tyr259 may act as an agonist binding site, offering a target for developing novel antifungal drugs against ascomycetes.
