Identification, genomic structure, and screening of the vacuolar proton-ATPase membrane sector-associated protein M8-9 gene within the COD1 critical region (Xp11.4).

PURPOSE: Our goal is to identify the gene responsible for X-linked cone-rod dystrophy (COD1) that has been localized to a limited region of Xp11.4. METHODS: A complete physical contig of the COD1 region was partially sequenced and subjected to BLAST searches to identify homologies with GenBank ESTs. ESTs were analyzed for overlapping or related cDNA sequences and retinal expression by PCR screening of multiple human retina cDNA libraries. RACE was performed to complete the missing 5' end of the transcripts. Transcripts were compared with genomic sequences to specify intron-exon boundaries. Genomic DNAs from COD1-affected males from 3 families were screened for mutations using direct PCR sequencing of the exons. RESULTS: The vacuolar proton-ATPase membrane sector-associated protein M8-9 (APT6M8-9) gene was identified within our critical region. We confirmed its retinal expression and its genomic location in our physical contig. Eight exons (with flanking intronic sequences) were characterized from partial cDNA sequence and genomic sequence data. An additional 5' end exon was identified using RACE. No mutations were found in the COD1-affected males. CONCLUSIONS: The combination of disease mapping and information from the Human Genome project has enabled us to identify candidate genes within the COD1 region, including APT6M8-9 gene. We found no evidence that this gene is responsible for COD1 in our families, but it may be an important candidate for other diseases that have been mapped to this region of the X chromosome.

Genomic structure of the mouse Ap3b1 gene in normal and pearl mice.

The mouse hypopigmentation mutant pearl is an established model for Hermansky-Pudlak syndrome (HPS), a genetically heterogenous disease with misregulation of the biogenesis/function of melanosomes, lysosomes, and platelet dense granules. The pearl (Ap3b1) gene encodes the beta3A subunit of the AP-3 adaptor complex, which regulates vesicular trafficking. The genomic structure of the normal Ap3b1 gene includes 25 introns and a putative promoter sequence. The original pearl (pe) mutation, which has an unusually high reversion rate on certain strain backgrounds, has been postulated to be caused by insertion of a transposable element. Indeed, the mutation contains a 215-bp partial mouse transposon at the junction point of a large tandem genomic duplication of 6 exons and associated introns. At the cDNA level, three pearl mutations (pearl, pearl-8J, and pearl-9J) are caused by deletions or duplications of a complete exon(s).

Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan.

Perlecan, the predominant basement membrane proteoglycan, has previously been shown to contain glycosaminoglycans attached at serine residues, numbers 65, 71, and 76, in domain I. However, the C-terminal domains IV and V of this molecule may also be substituted with glycosaminoglycan chains, but the exact substitution sites were not identified. The amino acid sequence of mouse perlecan reveals many ser-gly sequences in these domains that are possible sites for glycosaminoglycan substitution. We expressed recombinant domain IV and/or V of mouse perlecan in COS-7 cells and analyzed glycosaminoglycan substitution. Both heparan sulfate and chondroitin sulfate chains could be detected on recombinant domain V. One site, ser-gly-glu (serine residue 3593), toward the C-terminal region of domain V is a substitution site for heparan sulfate. When this sequence was absent, chondroitin/dermatan sulfate substitution was deleted, and the likely site for this galactosaminoglycan substitution was ser-gly-ala-gly (serine residue 3250) on domain V.

Identification of sites in domain I of perlecan that regulate heparan sulfate synthesis.

Perlecan is primarily a heparan sulfate containing proteoglycan found in all basement membranes. Rotary shadowed images of perlecan show it to contain three glycosaminoglycan (GAG) side chains extending from one end of its core protein. Domain I is at the N terminus of perlecan and contains three closely spaced Ser-Gly-Asp sequences that may serve in GAG attachment. We evaluated the serines in these three sequences for GAG attachment by preparing a cDNA construct encoding for the N-terminal half (domains I, II, and III) of perlecan and then a series of constructs containing deletions and mutations within domain I of the domain I/II/III construct, expressing these constructs in COS-7 cells, and then analyzing the recombinant product for GAG side chains and GAG type. The results showed that all three serine residues in the Ser-Gly-Asp sequences in domain I can accept both chondroitin and heparan sulfate side chains but that a cluster of acidic residues N-terminal to these sequences is the primary determinant responsible for targeting these sites for heparan sulfate. Furthermore, there are two elements that can enhance heparan sulfate synthesis at a targeted site: 1) the presence of a the SEA module in the C-terminal region of domain I and 2) the presence of multiple acceptors in close proximity. These results indicate that the proportion of heparan and chondroitin sulfate at any one site in domain I of perlecan is regulated by multiple factors.

Nuclear retinoic acid receptors in the lacrimal gland.

The lacrimal gland secretes and metabolizes retinoids and responds to retinoic acid in culture. Like other retinoid responsive organs it is expected to express the nuclear retinoid receptors. The goal of this study was to identify the retinoic acid receptors (RAR) in the lacrimal glands of rats, rabbits, and humans. Total RNA was prepared from whole lacrimal glands and rat lacrimal gland acinar cells grown in culture. RNA was subjected to Northern blot analysis and probed for the RAR alpha, RAR beta, and RAR gamma mRNAs. Nuclear extracts of rat and rabbit lacrimal glands were incubated with 3H-all-trans retinoic acid and analyzed by gel filtration chromatography. Western blots of the nuclear extracts were probed using monoclonal antibodies to RAR alpha and RAR beta. Rat lacrimal gland expresses RAR alpha mRNA with two transcripts (3.8 and 3.0 kb), a single RAR beta mRNA transcript (3.3 kb), and a single RAR gamma mRNA transcript (3.3 kb). Cultured rat lacrimal acinar cells also expressed the mRNA for all three RAR subtypes. Rabbit lacrimal glands express mRNAs for RAR alpha (3.7 and 2.9 kb) and RAR beta (3.2 kb) but RAR gamma mRNA is not detectable. Human lacrimal glands also express mRNA for RAR alpha (3.5 and 2.3 kb), RAR beta (3.4 kb) and RAR gamma (3.0 kb). Lacrimal gland nuclear extracts contain proteins in the 50 kDa range that specifically bind retinoic acid with Kd = 1.25 nM in rat lacrimal gland and 0.3 nM in rabbit. The monoclonal antibodies identified RAR alpha and RAR beta in both rat and rabbit lacrimal glands. The results of this study support a role for retinoids in maintaining the structure and function of the lacrimal gland. The presence of RARs suggests potential interactions of these receptors with other members of their superfamily, including androgen and thyroid receptors, which also may be involved in lacrimal function.

Simultaneous effects of the platelet 5-HT2 and alpha 2-adrenergic receptor populations on phosphoinositide hydrolysis.

The interaction of multiple receptor populations on a common second messenger system is a critical aspect of cell function and may be involved in pathology. We studied the interactions of the 5-HT2, alpha 2-adrenergic and prostaglandin (PGI2) receptors on phosphoinositide (PI) turnover in human platelets. Serotonin and epinephrine (EPI) stimulated PI hydrolysis in a dose-dependent manner. The PI turnover response to serotonin was mediated by the 5-HT2 receptor. The PI response to EPI was mediated by alpha 2-adrenergic receptors. An additive PI turnover response was generated by the combination of 5-HT and EPI. The sum of the maximal responses to 5-HT (72.5 +/- 4.9%) and EPI (56.0 +/- 4.2%) approximated the maximal response (129.3 +/- 9.5) to the combination. Prostacyclin (PGI2) at 1 microgram/mL reduced PI turnover by 21.8 +/- 1.1%. The PI response to 5-HT and EPI was not significantly altered once the reduction in the baseline PI turnover by PGI2 is taken into account. Similarly, PGI2 did not reduce PI hydrolysis stimulated by a combination of 5-HT (0.2 mM) and EPI (0.1 mM) once the decrease in baseline was taken into account (p greater than 0.20). The summation of serotonin stimulation of PI turnover by a combination of both epinephrine and serotonin was blocked by either yohimbine or ketanserin. These studies indicate: (1) the pool of phospholipases appears to exceed the maximal capacity of the individual alpha 2-adrenergic and 5-HT2 receptor populations to activate this second messenger system. (2) inhibition of serotonin or epinephrine-stimulated PI turnover by prostacyclin is due to a lowering of basal PI turnover. Future studies should examine other cell systems to assess the generalizability of these findings regarding the differences in effects on a second messenger system when activated by one receptor population as opposed to two different receptor types.