Class III myosins are one of many classes of myosin motor proteins. The first class III myosin, ninaC (neither inactivation nor afterpotential C), was identified in Drosophila as a gene responsible for abnormal phototransduction (Montell and Rubin 1988). This novel class of myosin is unique in having an N-terminal putative kinase domain joined to a myosin motor domain. The second class III myosin was isolated from the horseshoe crab Limulus polyphemus as a phosphoprotein regulated by a circadian clock; it is exclusively expressed in photoreceptors (Battelle et al. 1998). Subsequently, human class III myosin, MYO3A, was isolated by degenerated polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR from retina and a retinal pigment epithelial cell line (Dose and Burnside 2000). A shorter class III isoform, MYO3B, encoded by a different gene from MYO3A, was also identified (Dose and Burnside 2002).
Biochemical and Biophysical Properties
Given its sequence similarity to other myosins, it was predicted that class III myosins function as molecular motors, enzymes characterized by the ability to interact with actin to produce mechanical force by hydrolyzing ATP. Hicks et al. demonstrated that Drosophila myosin III in the soluble fraction of a retinal homogenate binds to actin in an ATP-sensitive manner (Hicks et al. 1996), although to date there has been no report demonstrating that purified Drosophila myosin III has ATPase activity. The sequence of the phosphate-binding loop or P-loop, known as a nucleotide-binding motif, consists of GESGAGKT in many myosins; however, the sequence of Drosophila myosin III is GESYSGKS. Indeed, myosin III from horseshoe crab binds actin, but lacks ATPase activity (Kempler et al. 2007). Amino acids which form a salt bridge, E459-R238 in the case of Dictiostelium myosin II, are essential for ATP hydrolysis. Horseshoe crab myosin III has H487 at the equivalent position for R238. The first experimental evidence to demonstrate that myosin III is an actual motor protein was reported in 2003 (Komaba et al. 2003). The authors showed that human myosin IIIA missing the tail domain has an ATPase activity with Vmax of 0.34 s−1 and is capable of gliding actin filament at 0.11 μm/s, which is relatively slower compared to many other myosins. Kinetic analysis demonstrated that the dephosphorylated motor domain of human myosin IIIA spends the majority of its time during the ATP hydrolysis cycle attached to actin suggesting that myosin III moves along actin cables without dissociating (Kambara et al. 2006).
Kinase activity in a class III myosin was originally observed in expressed myosin III from Drosophila, where the kinase domain phosphorylates various substrates including itself (Ng et al. 1996); however, the biochemical or physiological role of autophosphorylation of Drosophila myosin III remains unclear. Drosophila myosin III is phosphorylated in the p174 tail domain by protein kinase C and this phosphorylation is required for normal phototransduction, although the effect of phosphorylation on the biochemical properties of myosin III has not been shown (Li et al. 1998).
Horseshoe crab myosin III is also autophosphorylated and phosphorylated by protein kinase A (PKA) in loop 2, which is at the interface of actin binding (Kempler et al. 2007), suggesting that the interaction between myosin III and actin is modulated by cAMP-mediated phosphorylation and/or autophosphorylation.
Human myosin IIIA also undergoes autophosphorylation in the motor domain, which significantly reduces its affinity for actin (Kambara et al. 2006) resulting in a decrease in the duty ratio (Komaba et al. 2009). Deletion of the kinase domain affects both the biochemical and cell biological properties of myosin IIIA. A kinase domain-deletion mutant has a twofold higher Vmax and fivefold higher affinity for actin, compared to that of myosin IIIA with the kinase domain (Dose et al. 2008). Erickson et al. reported that striped bass myosin IIIA localizes to the cytoplasm and to the tip of filopodia in HeLa cells and that deletion of the kinase domain reduced cytoplasmic localization while enhancing localization at the tips of filopodia, suggesting that the kinase domain inhibits localization to filopodial tips (Erickson et al. 2003).
Deletion of Drosophila ninaC causes abnormal phototransduction and light- and age-dependent retinal degeneration (Porter et al. 1992). Deletion of the motor domain leads to a change in the subcellular distribution of myosin III and a phenotype indistinguishable from that of a null mutant, while mutations in the kinase domain result in defects in normal phototransduction, but no retinal degeneration (Porter and Montell 1993). The study using temperature-sensitive mutants demonstrated that the motor domain is required for maintenance of the retinal structure, but not for normal phototransduction (Porter and Montell 1993).
Myosin III is a major calmodulin-binding protein and the distribution of calmodulin in the retina of Drosophila is dependent on myosin III expression (Porter et al. 1993). In the mutant lacking p174, which is specifically localized to rhabdomeres, microvilli-packed structures in photoreceptor cells, calmodulin does not concentrate in rhabdomeres, whereas deletion of p132, the cell body–specific isoform, results in a decrease in cytoplasmic calmodulin.
Recent studies suggested that myosin III is involved in the transport of signaling molecules that undergo light-dependent translocation between the rhabdomere and the cell body in Drosophila photoreceptors. Upon light illumination, visual arrestin Arr2 translocates into rhabdomeres from the cell body and inactivates rhodopsin. Arr2 binds to myosin III mediated by phosphoinositides and this translocation is hindered in myosin III null and p132-lacking mutants (Lee and Montell 2004). However, another group demonstrated that light-dependent translocation of Arr2 does not require myosin III (Satoh and Ready 2005).
Gqα, an activator of phospholipase Cβ, translocates from the rhabdomere to the cell body in response to light stimulation and returns to the rhabdomere in the dark. The rate of translocation from the rhabdomere to the cell body in myosin III null mutants is similar to that of wild type; however, upon return to the dark, transport to the rhabdomere is significantly slower in myosin III mutants (Cronin et al. 2004).
Localization of transient receptor potential-like (TRPL) channels in the rhabdomere of myosin III mutants raised in the dark resembles that of wild type, whereas in orange light translocation to the cell body is partially inhibited in myosin III null mutants (Meyer et al. 2006).
One of the binding partners of myosin III in Drosophila is INAD, a scaffolding protein for signalplex consisting of phospholipase C, protein kinase C, and calmodulin. Disruption of binding between myosin III and INAD leads to defects in the termination of the photoresponse (Wes et al. 1999).
Vertebrate myosin IIIA is expressed at high levels in the retina and at lower level in the brain and testis. It localizes to the calycal processes of rod and cone photoreceptors (Dose et al. 2003).
Transgene expression of GFP-tagged myosin IIIA in Xenopus rod photoreceptors produces abnormal calycal processes and causes subsequent rod degeneration (Lin-Jones et al. 2004). Transgene-induced long and thick calycal processes contain a larger number of actin bundles, suggesting that myosin III regulates the organization of the actin-cytoskeleton in the rod photoreceptors.
Mutations in myosin III found in an Israeli family cause nonsyndromic progressive hearing loss, but have no effect on vision, although myosin III is strongly expressed in retina (Walsh et al. 2002). Myosin IIIA localizes at the tip of stereocilia, which are actin protrusions found on the sensory hair cells in the inner ear, in the thimble-like pattern (Schneider et al. 2006). Overexpression of GFP-tagged full-length myosin IIIA localizes similar to endogenous myosin IIIA and does not change stereocilia structure; however a mutant lacking the kinase domain accumulates at a higher level at the tip and leads to elongation of the stereocilia and bulging of the tip (Schneider et al. 2006). Myosin IIIA colocalizes with espin 1, an actin-bundling protein, at stereocilia tips (Salles et al. 2009). Espin 1 interacts with 3THD1 of myosin IIIA and when coexpressed with myosin IIIA produces longer stereocilia suggesting that myosin IIIA regulates the length of stereocilia together with espin 1. Normal vision and late-onset deafness in patients with myosin IIIA mutations suggests that myosin IIIB, which has 3THDI but not 3THDII compensates for myosin IIIA in photoreceptors and partially in the inner ear.
Myosin III is a unique myosin in that it has a kinase domain at the N-terminus of its motor domain. Physiological functions of class III myosins have not been fully uncovered, especially in vertebrates; however, the production of model animals such as myosin III knockout or transgenic mice will provide key information regarding the roles of myosin III.
- Dose AC, Hillman DW, Wong C, Sohlberg L, Lin-Jones J, Burnside B. Myo3A, one of two class III myosin genes expressed in vertebrate retina, is localized to the calycal processes of rod and cone photoreceptors and is expressed in the sacculus. Mol Biol Cell. 2003;14(3):1058–73.PubMedPubMedCentralCrossRefGoogle Scholar