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.