Copyright  1998 by the Genetics Society of America

Molecular Genetic Dissection of Mouse Unconventional -VA: Head Region Mutations

Jian-Dong Huang,* M. Jamie T. V. Cope,†,1 Valerie Mermall,‡ Marjorie C. Strobel,* John Kendrick-Jones,† Liane B. Russell,†† Mark S. Mooseker,‡,§,** Neal G. Copeland,* and Nancy A. Jenkins,* *ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, †MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom, ‡Department of Biology, §Department of Pathology, **Department of Cell Biology, Yale University, New Haven, Connecticut 06520, ††Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Manuscript received September 19, 1997 Accepted for publication December 23, 1997

ABSTRACT The mouse dilute (d) encodes unconventional myosin-VA (MyoVA). Mice carrying null alleles of dilute have a lightened coat color and die from a neurological disorder resembling ataxia and opisthotonus within three weeks of birth. Immunological and ultrastructural studies suggest that MyoVA is involved in the transport of melanosomes in melanocytes and smooth endoplasmic reticulum in cerebellar Purkinje cells. In studies described here, we have used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles that vary in their effects on coat color and the nervous system. Seven of these mutations mapped to the MyoVA motor domain and are reported here. Crystallo- graphic modeling and mutant expression studies were used to predict how these mutations might affect motor domain function and to attempt to correlate these effects with the mutant phenotype.

HE mouse dilute (d) locus encodes unconventional motor is used for the long-range transport of Tmyosin-VA (MyoVA). Mice homozygous for null SER from the cell body to the dendritic shaft. This mutations at dilute have a lightened coat color and die hypothesis is consistent with recent studies indicating from a neurological disorder resembling ataxia and that the movement of membranous involves opisthotonus (arching of the head and neck) within both - and -based motors and with cur- three weeks of birth. The pigment defect in dilute mice rent models suggesting that provide the does not result from abnormal pigment production. tracks for movement over long distances while actin Rather, the lightened coat color results from the irregu- filaments provide for movement within local regions of lar clumping of melanosomes within the perinuclear the cytoplasm (Atkinson et al. 1992; Langford 1995). regions of the melanocyte and the subsequent uneven It is also consistent with immunofluorescence and im- release of the granules into the hair shaft (Russell and munoelectron microscopy studies showing that MyoVA- Russell 1948). This phenotype is consistent with im- associated organelles are present on both microtubules munolocalization experiments suggesting that MyoVA and actin filaments (Evans et al. 1997). functions in melanosome transport and/or melano- During the past century hundreds of forward muta- Provance Wu some tethering ( et al. 1996; et al. 1997). tions to dilute have been identified. Most of these alleles The neurological defects of dilute appear to result were produced in large-scale mutagenesis screens, first in part from defects in smooth endoplasmic reticulum using ionizing radiations, which, in certain germ-cell (SER) transport. In both the dilute rat and the dilute stages, primarily make large deletions as well as other mouse, SER has been reported to be missing from the complex rearrangements, and later with chemicals such Dekker- dendritic spines of cerebellar Purkinje cells ( as ethylnitrosourea (ENU) which, when spermatogonia Ohno Takagishi et al. 1996; et al. 1996). Since SER is are treated, primarily make point mutations. The vast still present in the dendritic shaft, it has been proposed majority of these induced mutations [called dilute opis- that MyoVA is required only for the short-range trans- thotonus (dop)ordilute lethal (dl)] are homozygous lethal port of SER into the dendritic spine and that another and presumably represent null alleles (Russell 1971; Strobel et al. 1990). Four viable classes of alleles were also recovered and presumably represent hypomorphic Corresponding author: Nancy A. Jenkins, ABL-Basic Research Pro- alleles. The first class, called dilute (d), produces a light- gram, P.O. Box B, Bldg. 539, Frederick, MD 21702-1201. Email: [email protected] ened coat color but no neurological defect. The second x 1Present address: University of California, Rm. 401 Barker Hall, #3202 class, called dilute intermediate (d ), is the most common Berkeley CA 94720-3202. class. The coat color of dx mice is intermediate between

Genetics 148: 1951–1961 (April, 1998) 1952 J.-D. Huang et al. wild type and d mice, and dx mice are neurologically (Version 8) provided by Genetics Computer Group, University normal. The last two classes, called dilute neurological (dn) of Wisconsin (Madison). Point mutations, deletions and inser- xn tions in the cDNA can be easily detected by this method. and dilute intermediate neurological (d ), have a lightened Preparation of protein samples and electrophoresis: A small coat color and a neurological defect that either disap- amount of frozen brain or spleen was chipped off the organ pears as the mice age or is mild and persists throughout under liquid nitrogen and homogenized in 0.5 ml of 5% ice- life. These two classes are distinguished by coat color, cold trichloroacetic acid (TCA) with a hand-held homoge- which is like that of d (dn)ordx (dxn) mice, respectively. nizer. A 10 ␮l aliquot was removed and the protein concentra- n tion was determined by the BCA assay (Pierce, Rockford IL). In some cases, d mutations were originally classified as The TCA precipitates were pelleted by centrifugation at op op viable d alleles. To avoid confusion with the lethal d 12k ϫ g for 10 min, 4Њ; the pellets were washed with water, alleles, we consider them all dn mutations in this report. respun and brought up in sample buffer (25 mm tris base, 38 In studies described here, we have used an RT-PCR- mm glycine, 5% SDS, 5% beta-mercaptoethanol, 50% glycerol, -mg/ml bromophenol blue) for a final protein concen 1ف based sequencing approach to identify the mutations and tration of 1 mg/ml. Samples were loaded at 20 ␮g/lane onto responsible for 17 viable dilute alleles. We hoped that, 5–20% mini-gradient SDS-PAGE gels (Laemmli 1970), and by determining the nature and position ofthe mutations transferred (Towbin et al. 1979) to PVDF membranes (Bio- responsible for each dilue allele and by correlating this Rad, Hercules CA). Blots were stained with directed information with mutation phenotype, we could gain against the head domain of myosin V (produced by F. S. Espindola new insights into the functional domains of MyoVA. ). This was produced in rabbits using a bacterially expressed fusion protein of the chicken myosin V In these studies we focused primarily on ENU-induced head domain fused to maltose-binding protein (Espreafico et alleles since they are most likely to be caused by point al. 1992) and purified on an amylose affinity column. Maltose- mutations, which are a very informative class of muta- binding protein reactivity was removed from the antisera by tions for structure-function studies. Members of all four absorption to a maltose-binding protein column and then viable classes of dilute alleles were sequenced. In the affinity-purified on a column constructed with the original n xn fusion protein. The final antibody was used at 0.05 ␮g/ml. case of the d, d , and d alleles, some spontaneous and Blots were processed for chemiluminescence according to the radiation-induced alleles were also included since few manufacturer’s directions (Boehringer Mannheim, Mann- ENU-induced alleles from these classes were available heim, Germany). For head alleles: In some experiments, anti- for study. Seven of the mutations mapped to the MyoVA body against the tail domain of myosin V (Espreafico et al. head and are reported here. 1992) was also used at 1 ␮g/ml. Blots were stripped and stained for myosin VI (assumed to be unaffected by the MyoVA muta- tions) as a loading control with anti-myosin-VI antibody (Has- son and Mooseker 1994) used at 1 ␮g/ml. MATERIALS AND METHODS Quantification of myosin V: Blots were scanned with a 600 Mice: The seven alleles reported in this study were gener- dpi, 8 bit greyscale scanner (Microtek Lab Inc., Torrance CA); the relative amount of V and VI per lane was ated by mutagenesis of (101/Rl ϫ C3H/Rl) F1 hybrid mice at Oak Ridge National Laboratory, Oak Ridge, TN. Six of the determined with Image software (National Institute of Health, seven alleles are extinct and only frozen tissues were available Bethesda, MD). In order to control for small differences in gel for analysis. The Myo5a2ENURcc allele is maintained at the Na- loading or transfer efficiency, the relative amount of myosin VI tional Cancer Institute-Frederick Cancer Research and Devel- for each lane of a given tissue was determined and used to opment Center by crossing carriers to C57BL/6J-d v se/d v se mice. normalize the amount of myosin V per lane. Northern analysis: Total RNA was prepared from brain, spleen and skin of C57BL/6J, 101/Rl, C3H/Rl and dilute mu- tant mice by the RNAzol method (Tel-Test, Inc, Friendswood, RESULTS TX). RNA was poly(A) selected once using the mRNA Purifi- cation kit from Pharmacia (Piscataway, NJ). For Northern analy- Mutant characterization: Each of the viable dilute al- sis, the RNA was electrophoresed through a 0.8% agarose gel leles sequenced in these studies was characterized as andthen transferred to a Hybond-Nϩ membrane (Amersham, follows. First, DNA from each mutation in the homozy- Arlington Heights, IL) by standard methods (Ausubel et al. gous condition was analyzed by Southern blot hybridiza- 1997). Hybridizations and washes were performed according tion with at least ten different restriction enzymes to deter- to Church and Gilbert (1984). The dilute cDNAs (1-1682, 1549-3928, 2904-7156) corresponding to the head and tail mine if the allele contained a large structural alteration regions were mixed and used as probe (Mercer et al. 1991). in Myo5a that would compromise its use for structure/ RT-PCR sequencing analysis: Total RNA was reverse tran- function studies. None of these viable mutations con- scribed using SuperScript reverse transcriptase (Bethesda Re- tained large structural alterations (data not shown), con- search Laboratories, Gaithersburg, MD) and amplified by PCR sistent with the fact that most were chemically-induced (94Њ denature, 55Њ hybridization, 72Њ elongation, 40 cycles). The resulting cDNA fragments were isolated from DNA, oligo- in spermatogonia. Next, Northern analysis was done to nucleotides and nucleotides with Centricon-100 sizing col- ensure that all alleles produced at least some RNA (see umns (Amicon, Beverly, MA). Purified fragments were directly below). Mutant RNAs were then reverse transcribed and sequenced with PRISM Ready Reaction DyeDeoxy Terminator sequenced. RNA from two tissues, brain and skin or Cycle Sequencing Kit (Perkin Elmer, Norwalk, CT) and read- spleen, was sequenced for each mutation. These tissues ready sequence was obtained with an automated sequencer (ABI). Fragments were sequenced from both directions. Dif- express all of the known alternative splice forms of ferences between the mutant and wild-type sequences were Myo5a (see below). Complete sequencing of the 5487 identified with the GCG Sequence Analysis Software Package bp coding region of the brain-specific isoform of Myo5a Myo5a Motor Domain Mutations 1953

Figure 1.—Strategy for PCR amplification of the Myo5a cDNA. The top of the figure shows an agarose gel containing the Myo5a PCR amplification products. The middle of the figure shows the DNAfragments(openrectangles) amplified by different primer sets. Brain cDNA is used as a tem- plate. Note that each individual in the Myo5a coding region is amplified by at least two independent PCR reactions. The bottom of the figure contains a schematic of the Myo5a cDNA showing the location of the primers on the cDNA (1 to 12 forward and 2Ј to 14Ј reverse). Untranslated regions are shown as hatched rectangles and the coding region as a blackened rectangle. The sequences of the primers are as follows: (1) GGCA CGAGCCTAGGCGGGGGGCG, (2) TCACATCTTCGCAGTAGCTGAAG, (3) CACGAAGCAAGGAGGCAGCCCTATG, (4) CTAT GGATTTGAAACATTTGAAATA, (5) ACCAGAGCTATTTCAAGATGATGAG, (6) GGATAAATACCAGTTTGGTAAG, (7) GTCT GCAGGAAGAAATTGC, (8) GGAGGAGCGCTATGATGACCTC, (9) TGCGCCAGGTGCGCCTGCTTACC, (10) CTGCTGGCCCA GAACCTGCAGCTG, (11) CATCAACAATTAACAGCATC, (12) ACAGATGTTCTACATTGTGG, (2Ј) GGATCCATGTCACCCA TGTTCTG, (3Ј) CTAAAAGGTAAGTTCTCATATTG, (4Ј) GCCTGCAGCTTGGAGATGGGCTTG, (5Ј) GTTTGACATGCGGGGC TTCTCA, (6Ј) CTCGTGCGCTGATCCGGATGGTC, (7Ј) CTGAATAACAATCGTGGCAGCT, (8Ј) AGGCGACTGAACTCATTCA GAAGG, (9Ј) CCTCAAGATGAGGACTTCCTCC, (10Ј) TCCTGCTTTTCAAGTTGTTCCATC, (11Ј) CCACTATATTGCTTCAAA CAGTGC, (12Ј) GTACCTGATCTGCATGCCC, (13Ј) CATATTCTGAGGAGAAATGCG, (14Ј) GTATGCCACTTAAATGCAGCAC.

(or any of the other isoforms expressed in skin and and spleen. Skin and spleen have identical splice forms spleen) could be accomplished in as little as three days. (#1 and #2), although the relative amount of each splice The RT-PCR based sequencing strategy is described form varies in the two tissues. In some cases, skin was not in Figure 1. Oligonucleotide primers homologous to available for analysis and spleen samples were substituted. Myo5a cDNA were designed in such a way that any given Head region mutations: Among the 17 dilute muta- region of the coding sequences is amplified in at least tions for which mutation data were obtained, seven were two independent PCR reactions with different primer located in the MyoVA head (Table 1, Figure 3; and sets. Oligonucleotide primers were also used as sequenc- Huang et al. 1998). All were missense mutations, consis- ing primers. Following PCR amplification, the RT-PCR tent with their chemical mode of induction in spermato- products were used for direct sequencing as described gonia. Five of the mutations were ENU-induced. ENU in materials and methods. Point mutations, deletions is a direct alkylating agent that can ethylate DNA at many and insertions in the cDNA can easily be detected by sites (for review see Shibuya and Morimoto 1993). Three this method. Any potential mutations were confirmed types of ENU-induced lesions have been shown to cause by analyzing tissues from a second animal. In control direct base misincorporation by DNA polymerase and are experiments, wild-type cDNA was sequenced from three thus thought to be the most important mutagenic lesions. strains, C57BL/6J, C3H/Rl, and 101/Rl. All but one of These are O 6-ethylguanine that induces G:C to A:T transi- the mutations analyzed in these studies arose on one of tions, O4-ethylthymine that induces T:A to C:G transitions these three backgrounds. All three wild-type sequences and O 2-ethylthymine that induces T:A to A:T and T:A to were identical (data not shown). One mutation, which G:C transversions (for a review see Marker et al. 1997). did not map to the head region, arose on a strain closely Three of the five ENU-induced head region mutations related to C57BL/6J, C57BL/10SnJ (see accompanying presumably resulted from O 6 or O 2 alkylations, two being article by Huang et al. 1998). T:A to A:T transversions and one being a G:C to A:T All of the known alternatively spliced forms of Myo5a transition (Table 1). The other two head region mutations were also sequenced in these studies. As shown in Figure were induced by methylnitrosourea (MNU) (Myo5a1MNURe) 2, three different alternative splice forms of Myo5a can or nitrosoethylcarbamate (NEC) (Myo5a8PNECIII). While lit- be identified by PCR amplification and agarose gel elec- tle information is available regarding the mutational trophoresis. Each splice form was isolated from the gel specificity of these two chemicals, both chemicals gener- and directly sequenced. Brain has a unique splice form ated mutations that were similar to those induced by (#3) that is not observed in other tissues such as skin ENU (Table 1). 1954 J.-D. Huang et al.

Figure 2.—PCR amplifi- cation of the three alterna- tive splice forms of Myo5a. The structure of the three alternative splice forms of Myo5a are shown to the right of the figure. The capi- tal letters represent exons as described previously (Sep- erack et al. 1995). Since each alternatively spliced form codes for a slightly dif- ferent tail, it has been sug- gested that each tail allows MyoVA to bind and transport different cargoes in different cell types. The bold arrows represent the primer set used in RT-PCR amplification. An agarose gel containing the Myo5a amplification products from brain, skin, and spleen is shown to the left of the figure. The sequence of spliced forms #1 though #3 was determined by direct sequencing after gel purification of each amplified fragment.

Three of the four viable classes of dilute alleles were Expression levels: These head-region mutations could represented by these head-region mutations (Table 1). have an effect on MyoVA in two different ways. First, The one missing class was the d class, which was not they could alter amino acids critical for head function surprising given that only one of the viable mutations such as actin binding and ATP binding/hydrolysis. Sec- analyzed in these studies was from the d class. The head- ond, they could destabilize Myo5a mRNA or protein or region mutations were roughly equally distributed along prevent correct protein folding, resulting in reduced the length of the MyoVA head and there was no obvious protein levels. To determine if any of the mutations clustering of mutations with respect to phenotypic class affect Myo5a mRNA levels, brain RNA from the seven (Figure 3). The MyoVA head can thus be mutated in different head region mutations was characterized by such a way that only coat color or both coat color and Northern analysis (Figure 4) and Myo5a mRNA levels the nervous system are affected. While it is conceivable quantitated relative to a wild-type control (Table 1). that a head region mutation could affect the nervous Myo5a mRNA levels appeared to be altered by three system without affecting coat color, to date no dilute mutations, Myo5a18ENURw (130%), Myo5a48ENURd (79%), and alleles have been reported that affect only the nervous Myo5a8PNECIII (71%) (Table 1). These differences were, system. These results argue against a model where the however, small and may represent simple experimental MyoVA head contains a region(s) important for MyoVA variation. function in melanocytes and another region(s) impor- To determine if any of the mutations affected MyoVA tant for MyoVA function in neurons; they are more protein levels, extracts from mutant spleen or brain consistent with a simple model where the severity of the were characterized by Western analysis using antibodies mutation determines its ultimate phenotype; the least directed against the MyoVA head (Figure 5; Table 1) severe mutations being dx and the most severe being dn. as well as the MyoVA tail (data not shown) and the

TABLE 1 Head region mutations

Phenotypea Myo5a MyoVA Mutant Pigment Neurological RNA levelsc Protein levelsc class Allele name dilution impairment Mutagenb Percent Percent Mutation dx Myo5a48ENURd ϩϩ 0 ENU 79 3 (79)d C454G, H138Q dx Myo5a18ENURw ϩϩ 0 ENU 130 41 T1262A, Y408N dx Myo5a1MNURe ϩϩ 0 MNU 102 117 C1586T, P516S dxn Myo5a94ENURd ϩϩ ϩ ENU 105 73 G2016A, R659H dxn Myo5a8PNECIII ϩϩ ϩϩ NEC 71 105 T419A, Y127N dxn Myo5a2ENURcc ϩϩ to ϩϩϩ ϩϩϩϩ ENU 100 30 C219G, P60R dn Myo5a4ENURk ϩϩϩϩ ϩ ENU 92 9 T954A, M305K a The relative severity of the phenotype of the various alleles with respect to coat color and neurological behavior has been estimated; it ranges from unaffected (wild type), indicated by 0, to extreme, indicated by ϩϩϩϩ. b ENU, ethylnitrosourea; MNU, methylnitrosourea; NEC, nitrosoethylcarbamate. All mutations were derived from exposed spermatogonia. c Percent wild-type control. d See text. Myo5a Motor Domain Mutations 1955

Figure 3.—Schematic representation of MyoVA showing the approximate location of the MyoVA head region mutations. The different functional domains of MyoVA including the head, neck and tail are indicated. The head region of MyoVA shows extensive homology with other myosins, including the ATP and actin binding sites (Mercer et al. 1991; Espreafico et al. 1992). The neck region contains six imperfect tandem repeats (the IQ repeats) and is the site of light chain binding (Espreafico et al. 1992). The tail region consists of a coiled-coil region followed by a globular domain. The presence of this coiled-coil region suggests that MyoVA is a dimeric molecule and ultrastructure studies support this prediction (Cheny et al. 1993). Mutations indicated below the schematic diagram affect only coat color while mutations indicated above the schematic diagram affect both coat color and the nervous system. relative levels quantitated relative to an internal MyoVI Myo5a2ENURcc (30%), is a dxn class mutation; and a mutation control. Both antibodies are specific for MyoVA and do with one of the mildest phenotypes, Myo5a18ENURw (41%), not cross-react with the related MyoV protein, MyoVB is a dx class mutation (Table 1). These results suggest (V.M.andM.S.M.,unpublishedresults).Bothantibodies that the phenotype caused by these three mutations gave identical results, with the exception of the Myo5a48ENURd may be partly, but not exclusively, explained by these mutation (see below). MyoVA protein levels appeared reduced protein levels. Several other mutations do not significantly reduced by at least three mutations (Table show this association and presumably result from muta- 1 and see below). The magnitude of the effect was tions that affect protein function (Table 1). roughly proportional to the severity of the mutant phe- One mutation, Myo5a48ENURd, that showed relatively notype for these three alleles. The mutation with the normal MyoVA protein levels when probed with a tail most severe phenotype, Myo5a4ENURk (9%), is a dn muta- region antibody, showed greatly reduced protein levels tion; the mutation with the next most severe phenotype, when probed with a head region antibody (Figure 5; Table 1). While the head antibody was a polyclonal against a large protein domain (see materials and methods), myosin heads, like actin, are notoriously bad antigens and often induce only a limited immune re- sponse. It is possible, therefore, that the head antibody recognizes a single major epitope(s) that is located in the most N-terminal region of the head (aa 1-137) and that the Myo5a48ENURd mutation, which is located at aa138,

Figure 4.—Northern blot analysis of Myo5a mRNA from brain of homozygous viable dilute mutant mice. The three principal 7, 8, and 12 kb Myo5a transcripts are shown. The Figure 5.—Western blot analysis of MyoVA levels in homo- complex transcription pattern of Myo5a results in part from zygous viable dilute mutant mice. Extracts from wild-type and the differential use of 3Ј poly(A) addition signals (Mercer et mutant mice were made from brain (Myo5a2ENURcc, Myo5a8PNECIII, al. 1991). The three transcripts appear to have identicalcoding Myo5a4ENURk and Myo5a18ENURw)orspleen(Myo5a94ENURd, Myo5a48ENURd capacity. RNA from C57BL/6J wild-type brain was used as and Myo5a1MNURe) and probed with a head region antibody. control. Gapd (1.35 kb) was used as a loading control. MyoVI was used as a loading control. 1956 J.-D. Huang et al. disrupts this epitope(s). The Myo5a48ENURd mutation must, entrance to the ATP binding pocket (Figure 6). H138 however, retain significant activity since the mutant phe- is sandwiched within a tight hydrophobic cluster that notype is limited to a slight dilution of coat color. includes the side chains of Y100 and F140 (Figure 7c). Crystallographic modeling: The primary structure of The introduction of a glutamine into this cluster might all myosin heads are highly conserved (Cope et al. 1996). be expected to be highly disruptive. However, Myo5a48ENURd This conservation makes it possible to model the struc- has a relatively mild phenotype, indicating that this mu- tural consequences of mutations in one myosin head tation has been accommodated within the 3-D structure using the three dimensional co-ordinates reported for of the motor domain with only a slight loss in functionality. other myosin heads (Fisher et al. 1995; Rayment et al. The Myo5a18ENURw mutation is caused by a T1262A 1993b; Smith and Rayment 1995; Smith and Rayment transversion that introduces a missense mutation Y408N 1996; Xie et al. 1994). In the studies outlined below, we into the protein (Figures 6 and 7a). Y408 (Y434 in chick) have modeled the MyoVA head region mutations using is conserved in over 90% of all myosins, but is replaced the three-dimensional structure reported for the chicken by an H in two myosin I’s, namely rat Myr3 and human skeletal muscle conventional myosin II head (Rayment IC (Cope et al. 1996). Y408 is involved in a series of et al. 1993b). In these studies, the head region is defined conserved hydrophobic interactions with residues from as the S1 fragment, which refers to the proteolytic frag- the well-known highly conserved myosin sequence EA/ ment obtained when conventional myosin II is digested SFGNAKT, forming a hydrophobic pocket close to the with papain. Following trypsin digestion, this S1 frag- ATP binding site (see Figure 7a). An asparagine in this ment is further cleaved into the N-terminal 25, central position would be unable to hydrogen bond with the 50 and C-terminal 20 kDa fragments (colored green, backbone oxygen of F232 and would generate a buried red, and blue, respectively, in Figures 6 and 7). polar residue. One would predict that the net effect would Six out of the seven MyoVA head region mutations be to destabilize the ATP binding site. However, it is could successfully be modeled using this approach (Fig- interesting to note that Myo5a18ENURwR, like Myo5a48ENURd,is ures 6 and 7). The Myo5a2ENURcc mutation could not be a mild mutation (Table 1). Thus, changes in these highly evaluated in these studies because it results from a conserved residues seem to have relatively minor effects C219G transversion that introduces a missense mutation on the function of MyoVA. Nevertheless, a phenotype P60R into the protein. P60 is located within an N-termi- is observed. nal extension region that varies dramatically among the The Myo5a1MNURe mutation is due to a C1586T transi- various classes of myosins. In the chicken skeletal muscle tion that introduces a missense mutation P516S into the myosin II, this region forms a beta-barrel that makes myosin (Figures 6, 7d, and 7e). P516 is located in one of little contact with the rest of the motor domain (see the regions believed to be intimately involved in myosin- ,aa 500–530) (Rayment et al. 1993aف) Figure 6) while in other myosins, this region may be actin interaction truncated or even absent. The role of this region is not Figures 7d and 7e). This residue is conserved in about known. The severity of the Myo5a2ENURcc phenotype may 85% of known myosins, but in a number of myosin I’s reflect an important function for the MyoVA N-terminal it is replaced by an A or a hydrophobic residue. The extension or may result from the reduced MyoVA pro- mildness of the Myo5a1MNURe phenotype suggests that the tein levels observed in Myo5a2ENURcc mice (Table 1). How- mutant myosin is partially active, providing additional ever, reduced protein levels do not correlate consis- evidence that this region of the actomyosin interface tently with severity (see Myo5a18ENURw). may be far less specific than previously imagined (see The Myo5a48ENURd mutation results from a C454G trans- also Cope et al. 1996). version that introduces a missense mutation H138Q into The Myo5a94ENURd mutation arises from a G2016A tran- the protein. H138 (H154 in chick) is absolutely con- sition that introduces a missense mutation R659H into served in all known myosins (Cope et al. 1996). It may the myosin. R659 (R673 in chick) is absolutely conserved be important in stabilizing the transition state during in all myosins and lies close to the bottom of the nucleo- ATP hydrolysis since H138 is located in the helix at the tide binding pocket (Figures 6 and 7f). It was previously

Figure 6.—The positions of the six missense mutations in the MyoVA head modeled onto the three-dimensional structure of chicken pectoralis myosin II motor domain (Rayment et al. 1993b). Above is shown the “conventional” view of the motor domain, with the nucleotide binding pocket at the top and the actin binding region at the bottom left. Below is shown the same structure rotated approximately 180Њ about the horizontal axis. The locations of the six mutations are indicated by arrows, with the corresponding chicken residues as determined by multiple alignment (Cope et al. 1996) in parentheses. The side chains of these chicken residues are shown in cyan. The Myo5a2ENURcc (P60R) mutation could not be modeled since the N-terminal beta-barrel (shown in this figure in green and marked with an asterisk) is not present in MyoVA. The 20 kDa, 50 kDa and 25 kDa proteolytic subdomains are shown in green, red and blue, respectively. The sulfate occupying part of the nucleotide binding site is shown in yellow-green. For orientation, the inserts show the entire S1 structure with the regulatory light chain in magenta and the essential light chain in yellow-green. (Graphics were prepared using Midas software (University of California, San Francisco) on a Silicon Graphics Indigo II workstation, followed by annotation in Freehand (Aldus) on an Apple Macintosh.) Myo5a Motor Domain Mutations 1957 1958 J.-D. Huang et al.

Figure 7.—Closeup views of the six mutations mapped onto the chicken myosin II crystal structure illustrating their local environment: (a) Myo5a18ENURw (Y408N); (b) Myo5a4ENURk (M305K); (c) Myo5a48ENURd (H138Q) and Myo5a8PNECIII (Y127N); (d) and (e) Myo5a1MNURe (P516S); and (f) Myo5a94ENURd (R659H). In the center, an overall view of the myosin motor domain is shown as described in Figure 6. Panels b, d, and f are in approximately the same orientation as this central view, while panels a, c, and e are rotated for clarity. The colors of the motor domain are as described in Figure 6: the mutated side chains are cream; the other side chains are magenta; sulfur is shown in yellow-green; and the oxygens of the sulfate are in red. In this Figure, the amino acids are numbered according to their positions in the chicken myosin II protein and the corresponding mouse MyoVA amino acid is shown in parentheses. In panel e, the proximity of P543 to actin in the actomyosin interface determined by Rayment and colleagues (Rayment et al. 1993a) is shown. One actin monomer is in blue, another in green, and the myosin is in red, with P543 labeled. Only a few side chains have been included for reference as it is not possible to determine the precise interactions between the myosin and actin side chains. In panel f, a spacefilling model of the region around R673 shows the cream NH1 and NH2 atoms of this residue. These are close to the O4 of the sulfate occupying part of the ATPase site that is just visible in this view. (Graphics were prepared using Quanta (Molecular Simulations, Inc.) and Midas (UCSF) software on a Silicon Graphics Indigo II workstation, and Rasmol followed by annotation in Freehand (Aldus) on an Apple Macintosh.) identified as a crucial residue that might be involved in or hydrolysis. The severity of the Myo5a8PNECIII phenotype the release of the ␥-phosphate from the ATPase site supports this prediction (Table 1). It is interesting that (Cope et al. 1996). An R659H change would normally be the mutations of residues Y127 (Myo5a8PNECIII) and H138 considered a mild mutation. The Myo5a94ENURd phenotype (Myo5a48ENURd), which are adjacent in the crystal structure fits this prediction, it has a mild effect on both coat (Figures 6 and 7c), result in phenotypes of differing color and the nervous system (Table 1). severity, at least with respect to the nervous system. The Myo5a8PNECIII mutation results from a T419A trans- The Myo5a4ENURk mutation is due to a T954A transver- version that introduces a missense mutation Y127N into sion that introduces a missense mutation M305K into the protein. Y127 (Y143 in chick) is over 90% conserved the myosin. M305 (L331 in chick) is not conserved, but in all myosins and in the remainder it is replaced by an F. it usually is a hydrophobic residue. It is located in a Y127 is adjacent to the hydrophobic cluster containing helix running along the top of the upper 50 kDa subdo- H138,which is absolutely conserved (Figure 7c). A muta- main and is likely to be a buried hydrophobic residue, as tion in Y127 (especially to an N) might disrupt this helix, it is in the chicken skeletal myosin II structure (Figures 6 located at the mouth of the ATP binding site, and might and 7b). A lysine in this position introduces a larger, be expected to have a deleterious effect on ATP binding charged sidechain that should significantly disrupt the Myo5a Motor Domain Mutations 1959 folding of the upper 50 kDa domain, perhaps resulting the tail domain, that result in non-syndromic recessive in the very low protein levels and the very severe pheno- deafness (Liu et al. 1997; Weil et al. 1997). type, at least with respect to coat color, that is observed In patients with familial hypertrophic cardiomyopa- in Myo5a4ENURk mice (Table 1). thy (HCM), forty different mutations have also been identified in human conventional ␤-cardiac myosin (MYH7) (reviewed in Rayment et al. 1995). HCM is an DISCUSSION autosomal dominant inherited cardiac disease, charac- In the studies described here, we have used an RT- terized by left ventricular hypertrophy and markedly PCR-based sequencing approach to identify the muta- variable phenotypic expression (Epstein et al. 1992; tions responsible for 17 viable dilute alleles that vary in Fananapazir et al. 1993). It is the most common cause their effect on coat color and the nervous system. Seven of sudden death in otherwise healthy individuals. Sur- of the mutations represented missense mutations that prisingly, 33 of the 40 characterized MYH7 mutations mapped to the MyoVA head. Three of the four viable are located in the motor domain (Rayment et al. 1995; classes of dilute alleles were represented by these motor Vikstrom and Leinwand 1996). This may reflect the domain mutations. The one missing class was the d class, fact that HCM is a dominant disease and MYH7 muta- which was not surprising, given that only one of the tions cause disease through dominant negative effects. viable mutations analyzed in these studies was from the Dominant negative mutations may be easier to generate d class. The MyoVA head can thus be mutated in such in the head region. The 33 MHY7 head region muta- a way that only coat color or both coat color and the tions are clustered around four specific regions in the nervous system are affected. myosin head: (1) the actin binding interface; (2) the Mutations in only two other classes of mammalian ATP binding site; (3) the region that connects the two unconventional myosins have been reported. The mouse reactive cysteines, SH1 and SH2; and (4) the light-chain Snell’s waltzer (sv) mutation is caused by mutations in binding region. unconventional Myo6 (Avraham et al. 1995). Homozy- Similar to the results reported for Myo7a (MYO7A) gous sv mice have defects in the inner ear and are and MYH7,mostoftheMyo5a head mutations were lo- completely deaf. Only one mutation in the Myo6 cated near regions important for motor domain function has been reported. This mutation results from a small including actin-binding (Myo5a1MNURe) and ATP-binding/ intragenic deletion that truncates the protein at the hydrolysis (Myo5a48ENURd, Myo5a18ENURw, Myo5a8PNECIII, and head/neck junction (Avraham et al. 1995). Likewise, Myo5a94ENURd). However, two of the mutations were located the mouse shaker-1 (sh1) mutation is caused by muta- in regions not previously identified as being important tions in unconventional Myo7a (Gibson et al. 1995). for motor domain function. One mutation, Myo5a2ENURcc, Interestingly, homozygous sh1 mice have a phenotype was located in an N-terminal extension that is unique that is nearly identical to that of homozygous sv mice. to the MyoV class of unconventional myosins and is Three Myo7a mutations have been reported (Gibson et absent in other classes of myosins. The severity of the al. 1995), all being in the Myo7a head, the only region Myo5a2ENURcc mutation might indicate an important func- screened to date. One mutation is in a splice site, re- tion for this N-terminal extension; however, the reduced sulting in premature translation termination and would MyoVA protein levels observed in Myo5a2ENURcc mice make presumably be a null allele. The other two are arginine- this difficult to predict with certainty. The other muta- to-proline missense mutations, one of which is close tion, Myo5a4ENURk, by replacing a hydrophobic residue to the ATP-binding site and presumably affects ATP- with a charged basic residue, probably disrupts the fold- binding/hydrolysis or protein folding. ing of the upper 50 kDA subdomain. This disrupted Mutations in MYO7A have also been identified in folding could explain the very low levels of MyoVA pro- human type 1B patients (USH1B) tein observed in Myo5a4ENURk mice. (Weil et al. 1995). Usher syndrome type 1 is character- A unique feature of the Myo5a mutations analyzed ized by a profound congenital sensorineural hearing here is that they all are likely to encode with loss, constant vestibular dysfunction and prepubertal residual wild-type function. Such alleles are more likely onset of retinitis pigmentosa. Fourteen exons from the to identify important functional domains within a pro- head region of MYO7A have been screened for muta- tein than are null alleles, which often represent large tions in a total of 189 USH1B families (Weston et al. deletions or nonsense mutations within the protein cod- 1996). Out of 23 mutations, 13 were unique. Six of the ing sequence. This can easily be seen when one com- 13 mutations caused premature stop codons, 6 were pares the MYO7A head region mutations, which in many missense mutations, and 1 was a splicing defect. The 6 cases are null alleles, with the Myo5a mutations analyzed missense mutations were largely located in regions of here. While 6 of 13 MYO7A mutations truncate the the head thought to be involved in actin binding and protein in the head (Weston et al. 1996), all 7 Myo5a ATP binding/hydrolysis (Weston et al. 1996). Interest- mutations are missense mutations. ingly, three additional mutations in MYO7A have re- Another unique feature of the Myo5a mutations ana- cently been identified, two in the motor and one in lyzed here is that they can be grouped into multiple 1960 J.-D. Huang et al. different phenotypic classes and the nature and position R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl. John Wiley & Sons, New York. of the mutation can thus be correlated with the mutant Avraham, K. B., T. Hasson, K. P. Steel, D. M. Kingsley, L. B. phenotype. In this regard, it is interesting to note that Russell et al., 1995 The mouse Snell’s waltzer deafness gene the Myo5a head region mutations are roughly equally encodes an unconventional myosin required for structural integ- rity of inner ear hair cells. Nature Genet. 11: 369–375. distributed along the length of the MyoVA head and Cheney, R. E. K., M. K. O’Shea, J. E. Heuser, M. V. Coelho, J. 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