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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.

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