BACKGROUND
otopetrin <dig>  is the first described member of the otopetrin family, a novel gene family that encodes multi-transmembrane domain proteins.
the family was named for the conserved role of otop <dig> in the formation of otoconia and otoliths – "oto"  and "petros" .
otoconia are complex calcium carbonate biominerals in the utricle and saccule of the vertebrate inner ear that are required for the normal sensation of linear acceleration and gravity.
degeneration or displacement of otoconia can lead to vertigo and loss of balance  <cit> .
three mutant mice and one zebrafish model with mutations in otop <dig> have been described: tilted   <cit> ; mergulhador   <cit> ; inner ear defect   <cit> ; and backstroke   <cit> , respectively.
all of these mutants lack otoconia or otoliths, but have normal inner ear development.
in zebrafish, the morpholino knockdown of otop <dig> phenocopies the tlt mutation, showing otolith agenesis with no disruption of the patterning of the developing inner ear  <cit> .
the otopetrin family in most vertebrates studied consists of three genes clustered in two chromosomal locations: otop <dig>  and the paralogous tandem genes otop <dig> and otop <dig> .
vertebrate otopetrins share a conserved gene and protein structure, with no homology to other transporters, channels, exchangers, or receptors.
a preliminary secondary structure prediction based on the human, mouse, rat, zebrafish, and fugu protein sequences suggested a topology of ten transmembrane domains  with cytosolic amino and carboxy termini.
additionally, tblastn searches in the est and genomic databases identified regions of homology with the duf <dig> domain in a number of arthropod and nematode proteins.
duf <dig>  is a  <dig> amino-acid consensus sequence of unknown function that defines the duf <dig> family, with members in c. elegans and d. melanogaster.
the two regions of maximum homology with duf <dig> found in vertebrate otopetrins correspond to putative tm domains 3– <dig> and 9– <dig>  respectively, and were initially designated duf270-i and duf270-ii  <cit> .
here, we report a comparison of evolutionary constraint and hydropathy profile analysis of  <dig> vertebrate otopetrins and arthropod and nematode duf <dig> proteins, demonstrating that the genes that encode these proteins constitute a single family that we have renamed the otopetrin domain protein  gene family.
the refined topologic model of the odp proteins includes  <dig> putative tm domains clustered into three "otopetrin domains" , with a strong degree of sequence conservation across widely divergent groups of metazoa.
these regions of highest homology and evolutionary constraint, including the fyr box in the cytoplasmic tail, may represent important functional sub-domains.
biochemical studies in transfected cells show that otop <dig> modulates the manner in which cells handle intracellular calcium in response to purinergic stimuli  <cit> .
the lack of known functional domains, such as atp-binding domains, selectivity pores, or g-protein-binding consensus sequences, suggests that either the odp family has a novel function that significantly differs from the activities of known channels, transporters, or receptors, or that the odp genes encode novel functional motifs.
we hypothesize that these motifs would likely occur within the evolutionarily constrained regions, as has been shown for other well-conserved gene families  <cit> .
the challenge remains to define the functional domains of the odp family, with sequence and analyses reported here providing a step in that direction.
RESULTS
comparative sequence data set
the annotation of the otop <dig>  otop <dig>  and otop <dig> genes in the human, mouse, rat, zebrafish, and fugu genomes is described elsewhere  <cit> .
orthologous otopetrin sequences were generated using a targeted sequencing approach   or identified through tblastn searches of available whole-genome sequences.
the phylogenetic relationships of vertebrate otopetrin and arthropod and nematode duf <dig> genes were deduced from a total of  <dig> complete or nearly complete open reading frames in  <dig> species .
fragmentary, but clearly otopetrin-related, sequences were also identified in urochordates , echinoderms , and cnidarians , however were not complete enough to include in this analysis.
in some instances, the nucleotide accession number corresponds to a †scaffold, ††cosmid, or †††fosmid record; in those cases, the accession number of the otop or otopl annotation  is indicated in parenthesis.
* ensembl accession number
phylogenetic relationships and revised nomenclature of vertebrate otopetrins and arthropod and nematode duf <dig> genes
a maximum-likelihood phylogenetic tree was created from the multi-sequence alignment of each encoded protein .
the vertebrate, arthropod, and nematode sequences form distinct monophyletic groups, each containing three or more paralogous groups.
this arrangement suggests that the ancestral metazoan genome may have contained a single otopetrin-like gene, with subsequent duplications giving rise to the paralogs in the different phyla after the three lineages diverged.
based on the positions in the tree of the named mouse and human sequences, the three vertebrate paralogous groups correspond to otop <dig>  otop <dig>  and otop <dig>  otop <dig> and otop <dig> are more closely related to each other than either is to otop <dig>  a clustering that parallels the genomic organization of the otop genes in the vertebrate genomes.
the arthropod and nematode duf <dig> sequences, in which encoded proteins cluster independently in the tree from the vertebrate otopetrin sequences, have been renamed as otopetrin-like proteins , and the paralogous groups have been assigned arbitrary letters.
this is in agreement with the hugo gene nomenclature committee guidelines for gene families and grouping  <cit> .
like vertebrates, arthropods also have three paralogous groups of otopls.
the grouping in nematodes is more complex: there appears to be three major groups of otopls, as in vertebrates and arthropods, but each group itself contains two or more paralogous groups as a result of species-specific gene duplications.
in summary, vertebrate otopetrins and arthropod and nematode otopl genes have been grouped as a single family that we named collectively the otopetrin domain protein  gene family.
refined topological model for odp insertion into the lipid bi-layer
conserved primary sequence is indicative of an underlying conserved tertiary structure, and the evolutionary information contained in an alignment of related sequences can be leveraged to improve predictions of shared structures  <cit> .
we took advantage of the deep multi-sequence alignment and phylogenetic tree of the odp family to reexamine the predicted topology of the odps .
a hydropathy profile was generated that employs phylogenetic averaging  <cit>  on hydropathy scale values for amino acids  <cit>  to improve the detection of conserved hydrophobic regions, which might correspond to tm domains.
the hydropathy profile revealed  <dig> strong hydrophobic regions, ten of which overlap with the originally predicted tm domains  <cit> .
likewise, the memsat <dig>  <cit>  and tmap  <cit>  algorithms, which take into account leveraged evolutionary information, also predicted  <dig> tm helices for odp family members that overlap well with the constrained regions and hydrophobic regions in our profile .
the refined topological model for the odp family thus consists of  <dig> tm domains, with both the n- and c-termini in the cytosol, and in which the two newly identified tm domains are tm <dig> and tm <dig>  respectively.
as shown in figure 2b, there are three discrete regions with maximum evolutionary constraint among vertebrates, arthropods and nematodes, which we have designated otopetrin domain  -i, -ii, and -iii, respectively.
among the tm domains, tm <dig> and tm <dig> show the poorest conservation and evolutionary constraint across species.
on the other hand, the loops connecting the tm domains show little sequence conservation or evolutionary constraint, strongly suggesting that the tm domains are the primary functional regions of the odp family .
despite the poor loop sequence conservation, the number of amino acids in  <dig> of the  <dig> loops separating tm domains is highly conserved , suggesting that the spacing of most of the tm domains relative to one another may be important for the tertiary structure and function of odp family members.
of note, the length of loop  <dig>  within od-i, is highly variable across all phyla, but conserved in vertebrates , as are all other loops except for loop  <dig>
#number of amino-acid residues within the n-terminal, interloop, and c-terminal domains.
, inner loop; , outer loop; sd, standard deviation.
*incomplete n-terminal sequence data were excluded from n-terminal loop length calculations.
homology between otop and otopl sequences extends beyond the canonical duf <dig> domain
duf <dig>  is a  <dig> amino-acid consensus sequence of unknown function.
early tblastn-based database searches identified regions of homology with the duf <dig> domain in both vertebrate otop and arthropod and nematode otopl proteins  <cit> , now grouped together as the odp family.
inspection of the multi-species odp sequence alignment suggests that the homology among odp proteins extends beyond the canonical duf <dig> domain .
specifically, the n-terminal end of the duf <dig> consensus sequence can be extended to include three amino acids  that are conserved in most vertebrate  and nematode  odps examined.
at the c-terminal end, the amino-acid conservation continues well beyond the duf <dig> motif to include the entire c-terminal tail of vertebrate otop .
a 14-amino-acid consensus sequence for this highly conserved c-terminal tail, which we named the fyr box, is shown in figure  <dig>  the fyr box is a signature unique to the odp family, and is present in all odp proteins but not in any non-odp sequences in the databases of ests and non-redundant sequences.
CONCLUSIONS
comparative analyses of vertebrate otopetrins and arthropod and nematode otopl proteins revealed that they all share a tm domain structure and significant conservation of amino-acid sequence, suggesting that they constitute a single protein family, here renamed the odp family.
we have expanded the domains of homology to more accurately reflect the extent of sequence conservation between vertebrates, arthropods and nematodes, and have identified three evolutionarily constrained tm domain-rich areas that we have designated as otopetrin domains.
od-i and od-iii are the most highly conserved regions of the odp family.
tlt mice carry a missense mutation , which alters the hydrophobicity of the predicted tm <dig> domain within od-i, and leads to a presumed alteration in the membrane insertion or activity of otop <dig> and otoconial agenesis  <cit> .
the od-ii evolutionarily constrained region was not identified in the initial modeling, but mutations in otop <dig> within this conserved segment of the protein have been shown to cause otolith/otoconial agenesis in bks mutant fish   <cit>  and in mlh mutant mice   <cit>  , suggesting that this region is functionally important.
initial modeling of the otop proteins suggested a  <dig> tm domain model with cytosolic n- and c-termini  <cit> .
this model had several problems, including that sites consistent with the consensus sequence for n-glycosylation were predicted to be cytosolic.
the  <dig> tm domain model predicted by hydrophobicity and evolutionary constraint analysis places the proposed glycosylation sites in the extracellular space , and suggests that it may reflect a more accurate version of otop insertion into the lipid bilayer.
interestingly, the missense mutations in the tlt, mlh, and bks animal models, which lead to functional loss of otop <dig> activity, each occur within highly conserved transmembrane domains; such mutations often alter the hydrophobicity of the conserved tm domain, which may lead to alterations in the ability of the protein to insert and orient in membranes.
otop <dig> is required for the formation of vertebrate otoconia, a process that involves calcium carbonate biomineralization and requires the regulation of intracellular calcium.
biochemical studies in transfected cells show that otop <dig> modulates the manner in which cells handle intracellular calcium in response to purinergic stimuli  <cit> .
the mechanisms of calcium carbonate biomineralization are highly conserved in the development of otoconia and otoliths in the vertebrate inner ear, the formation of the avian eggshell, the mineralization of the arthropod exoskeleton, and the development of other mineralized structures such as the mollusk shell  <cit> .
there is evidence that some odp family members are expressed in tissues associated with calcium secretion and calcium carbonate-based mineralization.
in particular, ests from callinectes sapidus  reveal strong expression of the d. melanogaster otoplb ortholog in hypodermal tissues that are required for calcium mobilization during the mineralization of the chitinous exoskeleton  <cit> .
odp mrnas are also expressed in the hemocytes of various invertebrate species, which have been associated with the development of mineralized structures in mollusks  <cit> .
in mammals, otop <dig> is expressed in the lactating mammary gland  <cit> , perhaps functioning in the secretion of calcium into milk.
taken together, the sequence homology, structural constraint, and expression pattern suggest a conserved role for members of the odp family in the formation of mineralized structures.
further examination of odps and continued characterization of natural and induced mutations in these proteins through both physiologic and topologic studies may assist in better understanding the mechanisms of establishing and maintaining mineralized structures throughout the animal kingdom.

 otopetrin is the first described member of the otopetrin family, a novel gene family that encodes multi-transmembrane domain proteins. the family was named for the conserved role of otop in the formation of otoconia and otoliths – "oto" and "petros". otoconia are complex calcium carbonate biominerals in the utricle and saccule of the vertebrate inner ear that are required for the normal sensation of linear acceleration and gravity. degeneration or displacement of otoconia can lead to vertigo and loss of balance. three mutant mice and one zebrafish model with mutations in otop have been described: tilted; mergulhador; inner ear defect; and backstroke, respectively. all of these mutants lack otoconia or otoliths, but have normal inner ear development. in zebrafish, the morpholino knockdown of otop phenocopies the tlt mutation, showing otolith agenesis with no disruption of the patterning of the developing inner ear. the otopetrin family in most vertebrates studied consists of three genes clustered in two chromosomal locations: otop and the paralogous tandem genes otop and otop. vertebrate otopetrins share a conserved gene and protein structure, with no homology to other transporters, channels, exchangers, or receptors. a preliminary secondary structure prediction based on the human, mouse, rat, zebrafish, and fugu protein sequences suggested a topology of ten transmembrane domains with cytosolic amino and carboxy termini. additionally, tblastn searches in the est and genomic databases identified regions of homology with the duf domain in a number of arthropod and nematode proteins. duf is a amino-acid consensus sequence of unknown function that defines the duf family, with members in c. elegans and d. melanogaster. the two regions of maximum homology with duf found in vertebrate otopetrins correspond to putative tm domains 3– and 9– respectively, and were initially designated duf270-i and duf270-ii. here, we report a comparison of evolutionary constraint and hydropathy profile analysis of vertebrate otopetrins and arthropod and nematode duf proteins, demonstrating that the genes that encode these proteins constitute a single family that we have renamed the otopetrin domain protein gene family. the refined topologic model of the odp proteins includes putative tm domains clustered into three "otopetrin domains", with a strong degree of sequence conservation across widely divergent groups of metazoa. these regions of highest homology and evolutionary constraint, including the fyr box in the cytoplasmic tail, may represent important functional sub-domains. biochemical studies in transfected cells show that otop modulates the manner in which cells handle intracellular calcium in response to purinergic stimuli. the lack of known functional domains, such as atp-binding domains, selectivity pores, or g-protein-binding consensus sequences, suggests that either the odp family has a novel function that significantly differs from the activities of known channels, transporters, or receptors, or that the odp genes encode novel functional motifs. we hypothesize that these motifs would likely occur within the evolutionarily constrained regions, as has been shown for other well-conserved gene families. the challenge remains to define the functional domains of the odp family, with sequence and analyses reported here providing a step in that direction.  comparative sequence data set the annotation of the otop otop and otop genes in the human, mouse, rat, zebrafish, and fugu genomes is described elsewhere. orthologous otopetrin sequences were generated using a targeted sequencing approach or identified through tblastn searches of available whole-genome sequences. the phylogenetic relationships of vertebrate otopetrin and arthropod and nematode duf genes were deduced from a total of complete or nearly complete open reading frames in species. fragmentary, but clearly otopetrin-related, sequences were also identified in urochordates, echinoderms, and cnidarians, however were not complete enough to include in this analysis. in some instances, the nucleotide accession number corresponds to a †scaffold, ††cosmid, or †††fosmid record; in those cases, the accession number of the otop or otopl annotation is indicated in parenthesis. * ensembl accession number phylogenetic relationships and revised nomenclature of vertebrate otopetrins and arthropod and nematode duf genes a maximum-likelihood phylogenetic tree was created from the multi-sequence alignment of each encoded protein. the vertebrate, arthropod, and nematode sequences form distinct monophyletic groups, each containing three or more paralogous groups. this arrangement suggests that the ancestral metazoan genome may have contained a single otopetrin-like gene, with subsequent duplications giving rise to the paralogs in the different phyla after the three lineages diverged. based on the positions in the tree of the named mouse and human sequences, the three vertebrate paralogous groups correspond to otop otop and otop otop and otop are more closely related to each other than either is to otop a clustering that parallels the genomic organization of the otop genes in the vertebrate genomes. the arthropod and nematode duf sequences, in which encoded proteins cluster independently in the tree from the vertebrate otopetrin sequences, have been renamed as otopetrin-like proteins, and the paralogous groups have been assigned arbitrary letters. this is in agreement with the hugo gene nomenclature committee guidelines for gene families and grouping. like vertebrates, arthropods also have three paralogous groups of otopls. the grouping in nematodes is more complex: there appears to be three major groups of otopls, as in vertebrates and arthropods, but each group itself contains two or more paralogous groups as a result of species-specific gene duplications. in summary, vertebrate otopetrins and arthropod and nematode otopl genes have been grouped as a single family that we named collectively the otopetrin domain protein gene family. refined topological model for odp insertion into the lipid bi-layer conserved primary sequence is indicative of an underlying conserved tertiary structure, and the evolutionary information contained in an alignment of related sequences can be leveraged to improve predictions of shared structures. we took advantage of the deep multi-sequence alignment and phylogenetic tree of the odp family to reexamine the predicted topology of the odps. a hydropathy profile was generated that employs phylogenetic averaging on hydropathy scale values for amino acids to improve the detection of conserved hydrophobic regions, which might correspond to tm domains. the hydropathy profile revealed strong hydrophobic regions, ten of which overlap with the originally predicted tm domains. likewise, the memsat and tmap algorithms, which take into account leveraged evolutionary information, also predicted tm helices for odp family members that overlap well with the constrained regions and hydrophobic regions in our profile. the refined topological model for the odp family thus consists of tm domains, with both the n- and c-termini in the cytosol, and in which the two newly identified tm domains are tm and tm respectively. as shown in figure 2b, there are three discrete regions with maximum evolutionary constraint among vertebrates, arthropods and nematodes, which we have designated otopetrin domain -i, -ii, and -iii, respectively. among the tm domains, tm and tm show the poorest conservation and evolutionary constraint across species. on the other hand, the loops connecting the tm domains show little sequence conservation or evolutionary constraint, strongly suggesting that the tm domains are the primary functional regions of the odp family. despite the poor loop sequence conservation, the number of amino acids in of the loops separating tm domains is highly conserved, suggesting that the spacing of most of the tm domains relative to one another may be important for the tertiary structure and function of odp family members. of note, the length of loop within od-i, is highly variable across all phyla, but conserved in vertebrates, as are all other loops except for loop #number of amino-acid residues within the n-terminal, interloop, and c-terminal domains., inner loop;, outer loop; sd, standard deviation.*incomplete n-terminal sequence data were excluded from n-terminal loop length calculations. homology between otop and otopl sequences extends beyond the canonical duf domain duf is a amino-acid consensus sequence of unknown function. early tblastn-based database searches identified regions of homology with the duf domain in both vertebrate otop and arthropod and nematode otopl proteins, now grouped together as the odp family. inspection of the multi-species odp sequence alignment suggests that the homology among odp proteins extends beyond the canonical duf domain. specifically, the n-terminal end of the duf consensus sequence can be extended to include three amino acids that are conserved in most vertebrate and nematode odps examined. at the c-terminal end, the amino-acid conservation continues well beyond the duf motif to include the entire c-terminal tail of vertebrate otop. a 14-amino-acid consensus sequence for this highly conserved c-terminal tail, which we named the fyr box, is shown in figure the fyr box is a signature unique to the odp family, and is present in all odp proteins but not in any non-odp sequences in the databases of ests and non-redundant sequences.  comparative analyses of vertebrate otopetrins and arthropod and nematode otopl proteins revealed that they all share a tm domain structure and significant conservation of amino-acid sequence, suggesting that they constitute a single protein family, here renamed the odp family. we have expanded the domains of homology to more accurately reflect the extent of sequence conservation between vertebrates, arthropods and nematodes, and have identified three evolutionarily constrained tm domain-rich areas that we have designated as otopetrin domains. od-i and od-iii are the most highly conserved regions of the odp family. tlt mice carry a missense mutation, which alters the hydrophobicity of the predicted tm domain within od-i, and leads to a presumed alteration in the membrane insertion or activity of otop and otoconial agenesis. the od-ii evolutionarily constrained region was not identified in the initial modeling, but mutations in otop within this conserved segment of the protein have been shown to cause otolith/otoconial agenesis in bks mutant fish and in mlh mutant mice, suggesting that this region is functionally important. initial modeling of the otop proteins suggested a tm domain model with cytosolic n- and c-termini. this model had several problems, including that sites consistent with the consensus sequence for n-glycosylation were predicted to be cytosolic. the tm domain model predicted by hydrophobicity and evolutionary constraint analysis places the proposed glycosylation sites in the extracellular space, and suggests that it may reflect a more accurate version of otop insertion into the lipid bilayer. interestingly, the missense mutations in the tlt, mlh, and bks animal models, which lead to functional loss of otop activity, each occur within highly conserved transmembrane domains; such mutations often alter the hydrophobicity of the conserved tm domain, which may lead to alterations in the ability of the protein to insert and orient in membranes. otop is required for the formation of vertebrate otoconia, a process that involves calcium carbonate biomineralization and requires the regulation of intracellular calcium. biochemical studies in transfected cells show that otop modulates the manner in which cells handle intracellular calcium in response to purinergic stimuli. the mechanisms of calcium carbonate biomineralization are highly conserved in the development of otoconia and otoliths in the vertebrate inner ear, the formation of the avian eggshell, the mineralization of the arthropod exoskeleton, and the development of other mineralized structures such as the mollusk shell. there is evidence that some odp family members are expressed in tissues associated with calcium secretion and calcium carbonate-based mineralization. in particular, ests from callinectes sapidus reveal strong expression of the d. melanogaster otoplb ortholog in hypodermal tissues that are required for calcium mobilization during the mineralization of the chitinous exoskeleton. odp mrnas are also expressed in the hemocytes of various invertebrate species, which have been associated with the development of mineralized structures in mollusks. in mammals, otop is expressed in the lactating mammary gland, perhaps functioning in the secretion of calcium into milk. taken together, the sequence homology, structural constraint, and expression pattern suggest a conserved role for members of the odp family in the formation of mineralized structures. further examination of odps and continued characterization of natural and induced mutations in these proteins through both physiologic and topologic studies may assist in better understanding the mechanisms of establishing and maintaining mineralized structures throughout the animal kingdom. 
otop is necessary for the formation of otoconia and otoliths, calcium carbonate biominerals within the inner ear of mammals and teleost fish that are required for the detection of linear acceleration and gravity. our studies suggest a refined topologic model for odp insertion into the lipid bilayer of transmembrane domains, and highlight conserved amino-acid residues that will aid in the biochemical examination of odp family function. signature features of odp proteins are three "otopetrin domains" that are highly conserved between vertebrates, arthropods and nematodes, and a highly constrained predicted loop structure. the high degree of sequence and structural similarity of the odp proteins may suggest a conserved role in the intracellular trafficking of calcium and the formation of biominerals. multi-species comparison of the predicted primary sequences and predicted secondary structures of vertebrate otopetrin, and arthropod and nematode duf proteins, has established that the genes encoding these proteins constitute a single family that we renamed the otopetrin domain protein gene family.
 otopetrin encodes a multi-transmembrane domain protein with no homology to known transporters, channels, exchangers, or receptors. otop is necessary for the formation of otoconia and otoliths, calcium carbonate biominerals within the inner ear of mammals and teleost fish that are required for the detection of linear acceleration and gravity. vertebrate otop and its paralogues otop and otop define a new gene family with homology to the invertebrate domain of unknown function genes.  multi-species comparison of the predicted primary sequences and predicted secondary structures of vertebrate otopetrin, and arthropod and nematode duf proteins, has established that the genes encoding these proteins constitute a single family that we renamed the otopetrin domain protein gene family. signature features of odp proteins are three "otopetrin domains" that are highly conserved between vertebrates, arthropods and nematodes, and a highly constrained predicted loop structure.  our studies suggest a refined topologic model for odp insertion into the lipid bilayer of transmembrane domains, and highlight conserved amino-acid residues that will aid in the biochemical examination of odp family function. the high degree of sequence and structural similarity of the odp proteins may suggest a conserved role in the intracellular trafficking of calcium and the formation of biominerals. 