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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02424

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PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa

Raphaël Etournay^1,*, Aziz El-Amraoui^1,*, Amel Bahloul¹, Stéphane Blanchard¹, Isabelle Roux¹, Guillaume Pézeron¹, Nicolas Michalski¹, Laurent Daviet², Jean-Pierre Hardelin¹, Pierre Legrain³ and Christine Petit^1,

¹ Unité de Génétique des Déficits Sensoriels, INSERM U587, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France
² Hybrigenics, 3-5 impasse Reille, 75014 Paris, France
³ Département de Biologie Joliot-Curie, CEA, 91191 Gif-sur-Yvette, France

View larger version (32K): [in a new window]   Fig. 1. Myosin 1c binds to PHR1. (A) Predicted structure of myosin 1c. The position of the yeast two-hybrid bait is indicated. (B) Predicted structure of the four PHR1 isoforms. PHR1a, the longest isoform, contains an N-terminal 19 amino acid peptide, a pleckstrin homology (PH) domain, the E7 domain encoded by the alternatively spliced exon 7, a juxtamembrane domain (JMD) that displays no significant similarity to any known protein, a transmembrane (TM) domain, and six putative extracellular residues (CT6). Numbers indicate amino acid positions (1-243) according to the PHR1a sequence. The overlapping cDNAs encoding the PHR1 fragments found to interact with the myosin 1c tail in the yeast two-hybrid system are shown. They all lack the E7 domain. (C) Co-immunoprecipitation of PHR1 and the myosin 1c tail. Extracts from cotransfected HEK293 cells producing both the GFP-tagged PHR1a (lane S1) and the myc-tagged myosin 1c IQ4 tail (lane S2) were used. The proteins were co-immunoprecipitated by the antibody to myc (lane IP2). Extract from transfected cells producing GFP-PHR1 and the c-myc tag alone (lane S1) was used as a negative control (lane IP1). (D-F) In vitro binding assays. Mapping of the binding site of PHR1. Different fusion proteins shown in D were incubated with immobilised GST-tagged myosin 1c tail fragments (myo1c IQ4 tail, aa 762-1028; and myo1c T701, aa 701-1028) or with GST. (E) The GST-myosin 1c IQ4 tail binds to PHR1b and PHR1b.mid, but not to PHR1.JMD and PHR1a.AP. (F) Coomassie stained gel. GST-myosin 1c T701 binds to PHR1aC, PHR1bC, and PHR1a.PH. By contrast, it does not bind to the PH domain of ßV spectrin (ßV.PH). Positions of molecular mass markers are indicated in kDa on the right-hand side of blots.

View larger version (34K): [in a new window]   Fig. 2. Effect of Ca2+ and calmodulin on the myosin 1c-PHR1 interaction. (A) Effect of the Ca2+ concentration. Myosin 1c IQ4 tail or GST alone was incubated with [35S]PHR1b in the presence of 100 µM EGTA or 50 µM Ca2+. PHR1b interacts with the tail of myosin 1c in both cases (upper panel). In the reciprocal experiment, full-length myosin 1c binds to GST-PHR1b in the presence of 100 µM EGTA or 50 µM Ca2+ (lower panel). (B) Effect calmodulin (CaM). GST-PHR1b binds to 35S-labelled full-length myosin 1c in the absence (–) or presence of calmodulin (1.5, 7.5 or 15 µM) (upper panel). In the reciprocal experiment, GST-myo1c T701 or GST was incubated with 35S-PHR1b in the absence or presence of calmodulin (1.5, 7.5 or 15 µM). The myosin 1c IQ4 tail-PHR1b interaction is not affected by any of the calmodulin concentrations tested (lower panel). Positions of molecular mass markers are indicated in kDa on the right-hand side of blots.

View larger version (46K): [in a new window]   Fig. 3. Myosin VIIa binds to PHR1. (A) Predicted structure of myosin VIIa. The yeast two-hybrid myosin VIIa prey obtained using PHR1b as the bait is indicated. (B) Binding of the myosin VIIa tail to GFP-tagged PHR1a in cotransfected HEK293 cells. Protein extract from cotransfected HEK293 cells producing both the GFP-tagged PHR1a and the untagged-myosin VIIa tail (lane S2) was used for immunoprecipitation. The myosin VIIa tail and PHR1a are co-immunoprecipitated by the anti-GFP antibody (lane IP2). Extract from cotransfected cells expressing the myosin VIIa tail and GFP alone (lane S1) was used as a negative control (lane IP1). (C) GST-PHR1b binds to the 35S-labelled myosin VIIa prey fragment, entire myosin VIIa tail, and full-length myosin VIIa. The PHR1-myosin VIIa tail interaction is affected by neither the absence of Ca2+ (–), nor high free Ca2+ concentrations (10, 50 or 500 µM). (D) In the reciprocal experiment, GST-tagged myosin VIIa prey fragment or GST alone was incubated with in vitro translated [35S]PHR1b, [35S]PHR1b.mid or [35S]PHR1.JMD (see Fig. 1D). GST-myo7a prey binds to PHR1b and PHR1b.mid, but not to PHR1.JMD. (E,F) The myosin VIIa tail constructs (E) used for the in vitro binding assays in F are given a number (circled). GST-PHR1b or GST was incubated with different 35S-labelled myosin VIIa protein fragments. GST-PHR1b binds to Myo7a/SH3 MyTH4 FERM and Myo7a/MyTH4 FERM, but not to Myo7a/SH3 MyTH4. Binding is not detected with the MyTH4 FERM C-terminal tail fragment of myosin XVa (Myo15a/MyTH4 FERM). MyTH4, myosin tail homology 4; FERM, 4.1, ezrin, radixin, moesin; SH3, src homology 3. Positions of molecular mass markers are indicated in kDa on the right-hand side.

View larger version (40K): [in a new window]   Fig. 4. PHR1 can form dimers. (A) Yeast two-hybrid PHR1 preys obtained using PHR1a as bait. (B) PHR1 isoforms can form heteromers. Top panel, PHR1 constructs used for the in vitro binding assay. Middle panel, GST-PHR1a or GST alone was incubated with full length [35S]PHR1a, [35S]PHR1b, or [35S]PHR1d. GST-PHR1a interacts with every PHR1 isoform. Lower panel, PHR1.JMD is sufficient for PHR1 homomerisation. (C) Gel filtration analysis of the purified PH domains of PHR1a (PHR1a.PH) and ßV spectrin (ßV spectrin.PH). The figure shows the regression curve between the molecular mass and the partition coefficient (Kav) defined by Kav = (Ve – Vo)/(Vt – Vo), where Ve is elution volume of the protein, Vo, column void volume and Vt, total bed volume. The molecular masses of eluted fractions are estimated according to the fractions corresponding to four molecular mass standards: bovine serum albumin (BSA), ovalbumin, ribonuclease A and chymotrypsinogen A. PHR1a.PH is eluted as a dimer, whereas ßV spectrin.PH is eluted as a monomer. (D) Schematic diagram illustrating how a PHR1 dimer can recruit two myosin 1c, two myosin VIIa, or one myosin 1c and one myosin VIIa molecule to the plasma membrane.

View larger version (33K): [in a new window]   Fig. 5. Schematic representation of an inner ear sensory cell and proposed molecular model of the slow adaptation process. (A) The hair bundle is composed of 20-300 actin-filled stiff microvilli, called the stereocilia, arranged in three to four rows of increasing height. Stereocilia are held together by different types of lateral links. In addition, a single tip link joins the tip of each stereocilium to the lateral side of its taller neighbour in the adjacent row. (B) According to the gating spring hypothesis (Howard and Hudspeth, 1987), deflection of the hair bundle in the excitatory direction exerts a tension force on the tip links. The force is transmitted to the mechanoelectrical transduction (MET) channels (believed to be located close to the tip link insertion in the membrane), increasing their probability of being open. An influx of cations (mainly K+ ions, but also Ca2+ ions) through the open MET channels depolarises the hair cell leading to neurotransmitter release and signalling to the central nervous system via afferent nerve fibres. (C) A model of the slow adaptation process evoked by sustained deflection of the hair bundle. The Ca2+ influx through the open MET channels triggers the adaptation process (see Fettiplace and Ricci, 2003; Gillespie and Cyr, 2004). The increase of the stereociliar Ca2+ concentration weakens calmodulin (CaM) binding to the myosin 1c IQ motifs, which in turn interact with anionic phospholipids [such as PtdIns(4,5)P2 (PIP2)] in the membrane. This also leads to the dissociation of myosin molecules from actin filaments. The resulting reduction in the tension exerted on the stereocilia membrane is thought to underlie the slow adaptation process, i.e. a decrease of the MET channel open probability while the mechanical stimulus is persisting. The PHR1-myosin complexes are expected to function as elastic molecular crosslinkers that contribute to and modulate the membrane tension. Unlike myosin 1c-phospholipids interactions, they may not be dependent on the local Ca2+ concentration.

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