Mutational analysis of the Aspergillus ambient pH receptor PalH underscores its potential as a target for antifungal compounds.
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.
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain.
Daniel
D
Lucena-Agell
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain.
América
A
Hervás-Aguilar
Section of Microbiology, Imperial College London, Flowers Building, Armstrong Road, London, SW7 2AZ, UK.
Tatiana
T
Múnera-Huertas
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain.
Olga
O
Pougovkina
Section of Microbiology, Imperial College London, Flowers Building, Armstrong Road, London, SW7 2AZ, UK.
Joanna
J
Rudnicka
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain.
Antonio
A
Galindo
Section of Microbiology, Imperial College London, Flowers Building, Armstrong Road, London, SW7 2AZ, UK.
Joan
J
Tilburn
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain.|Section of Microbiology, Imperial College London, Flowers Building, Armstrong Road, London, SW7 2AZ, UK.
Herbert N
HN
Arst
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid, 28040, Spain. penalva@cib.csic.es.
Miguel A
MA
Peñalva
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D019393:Arrestin / Q000235:genetics / Q000378:metabolism; D001233:Aspergillus nidulans / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D058990:Molecular Targeted Therapy; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism
D000935:Antifungal Agents; D019393:Arrestin; D005656:Fungal Proteins; D008565:Membrane Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D025801:Ubiquitin