Interaction of protocadherin-15 with the scaffold protein whirlin supports its anchoring of hair-bundle lateral links in cochlear hair cells

Abstract

The hair bundle of cochlear hair cells is the site of auditory mechanoelectrical transduction. It is formed by three rows of stiff microvilli-like protrusions of graduated heights, the short, middle-sized, and tall stereocilia. In developing and mature sensory hair cells, stereocilia are connected to each other by various types of fibrous links. Two unconventional cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23), form the tip-links, whose tension gates the hair cell mechanoelectrical transduction channels. These proteins also form transient lateral links connecting neighboring stereocilia during hair bundle morphogenesis. The proteins involved in anchoring these diverse links to the stereocilia dense actin cytoskeleton remain largely unknown. We show that the long isoform of whirlin (L-whirlin), a PDZ domain-containing submembrane scaffold protein, is present at the tips of the tall stereocilia in mature hair cells, together with PCDH15 isoforms CD1 and CD2; L-whirlin localization to the ankle-link region in developing hair bundles moreover depends on the presence of PCDH15-CD1 also localizing there. We further demonstrate that L-whirlin binds to PCDH15 and CDH23 with moderate-to-high affinities in vitro. From these results, we suggest that L-whirlin is part of the molecular complexes bridging PCDH15-, and possibly CDH23-containing lateral links to the cytoskeleton in immature and mature stereocilia.

Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography

“…PCDH15 assembles in the membrane together with lipoma HMGIC Fusion Partner-Like 5 (LHFPL5,TMHS) (Ge et al, 2018, p. 5) and pull-down studies using protein fragments suggest that PCDH15 may also interact with TMC-1 (Maeda et al, 2014). PCDH15 is also theorized to interact with the cytoskeleton via whirlin (Michel et al, 2020), while TMC may be coupled to the cytoskeleton via CIB2 and ankyrin repeats (Tang et al, 2020). However, the molecular composition of the MT machinery and the 3D arrangements of each component remain unknown.…”

Deciphering the Molecular Interaction Between the Adhesion G Protein-Coupled Receptor ADGRV1 and its PDZ-Containing Regulator PDZD7

“…In harmonin, this extension folds as a hairpin followed by a short α-helix mediating the formation of a heterotopic supramodule between the first PDZ and the upstream Harmonin Homology Domain (HHD) and forming direct contacts with residues upstream of the partner’s PBM ( Yan et al, 2010 ) (PDB entry 3k1r). This supramodule displays ∼10 fold increased affinity for protocadherin-15 compared to the isolated PDZ ( Michel et al, 2020 ). In whirlin, the first and second PDZ domains interact via their respective C-terminal extensions, structured as hairpins, promoting the formation of a homotypic supramodule that improves the binding capacity of the PDZ tandem by ∼3 fold ( Delhommel et al, 2017 ).…”

Selective binding and transport of protocadherin 15 isoforms by stereocilia unconventional myosins in a heterologous expression system

“…All three main PCDH15isoforms; PCDH15-CD1, -CD2, and -CD3, which differ in their cytoplasmic domain (Fig. 1b), are expressed in the hair cell bundle 25,27 . We obtained or cloned murine full-length (FL) and generated cytoplasmic (cyto) domain cDNA constructs for all three isoforms.…”

“…Hair cells express three isoforms of PCDH15 (PCDH15-CD1, -CD2, and -CD3) that share a common extracellular domain but differ in the C-terminal region of their cytoplasmic tails 25 . The expression levels, localization, and role of each isoform is not fully understood but several studies have shown partial redundancy within the three PCDH15 isoforms during development with the CD2 isoform being essential for proper MET function in mature auditory hair cells [26][27][28] .…”