Hypochondroplasia (MIM 146000) is an autosomal dominant skeletal dysplasia with skeletal features similar to but milder than those seen in achondroplasia. Within the past year, the achondroplasia locus has been mapped to 4p 16.3 (refs 5-7) and mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have been identified in patients with the disorder. More than 95% of 242 cases reported so far are accounted for by a single Gly380Arg mutation. McKusick et al. proposed that achondroplasia and hypochondroplasia are allelic based on the similarities in phenotype between the two disorders and the identification of a severely dwarfed individual whose father had achondroplasia and whose mother had hypochondroplasia. There is also genetic linkage evidence that hypochondroplasia and achondroplasia map to the same locus. We therefore began a systematic screening of FGFR3 to detect mutations in patients with hypochondroplasia. We now report a single FGFR3 mutation found in 8 out of 14 unrelated patients with hypochondroplasia. This mutation causes a C to A transversion at nucleotide 1620, resulting in an Asn540Lys substitution in the proximal tyrosine kinase domain.

Desmoplakin (DP), plakoglobin (PG), and plakophilin 1 (PP1) are desmosomal components lacking a transmembrane domain, thus making them candidate linker proteins for connecting intermediate filaments and desmosomes. Using deletion and site-directed mutagenesis, we show that remarkably, removal of ∼1% of DP's sequence obliterates its ability to associate with desmosomes. Conversely, when linked to a foreign protein, as few as 86 NH2-terminal DP residues are sufficient to target to desmosomes efficiently. In in vitro overlay assays, the DP head specifically associates with itself and with desmocollin 1a (Dsc1a). In similar overlay assays, PP1 binds to DP and Dsc1a, and to a lesser extent, desmoglein 1 (Dsg1), while PG binds to Dsg1 and more weakly to Dsc1a and DP. Interestingly, like DP, PG and PP1 associate with epidermal keratins, although PG is considerably weaker in its ability to do so. As judged by overlay assays, the amino terminal head domain of type II keratins appears to have a special importance in establishing these connections. Taken together, our findings provide new insights into the complexities of the links between desmosomes and intermediate filaments (IFs). Our results suggest a model whereby at desmosome sites within dividing epidermal cells, DP and PG anchor to desmosomal cadherins and to each other, forming an ordered array of nontransmembrane proteins that then bind to keratin IFs. As epidermal cells differentiate, PP1 is added as a molecular reinforcement to the plaque, enhancing anchorage to IFs and accounting at least partially for the increase in numbers and stability of desmosomes in suprabasal cells.

A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 Ϯ 0.142 mm and 0.64 Ϯ 0.08 mm, and has an estimated 24.7 Ϯ 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an "in vitro" substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions.The vacuole is a reservoir for calcium (Machlon, 1984) and consequently plays an important role in calcium homeostasis (Miller et al., 1990;Allen and Sanders, 1995;Sanders et al., 1999). Regulation of vacuole calcium levels is complex involving a variety of calcium channels and pumps (Sanders et al., 1999;Sze et al., 2000). Sustained elevated levels of cytosolic calcium can be toxic (Hepler and Wayne, 1985), so under normal conditions, cytosolic calcium levels increase only transiently. Proteinaceous calcium buffers may serve as homeostats to attenuate the signal transduction system. Well-characterized protein calcium buffers include calreticulin and calsequestrin (Ostwald and MacLennon, 1974;Campbell et al., 1983b). Homologs of calsequestrin (Krause et al., 1989;Xing et al., 1994), calreticulin (Chen et al., 1994;Napier et al., 1995;Nelson et al., 1997), and calnexin (Li et al., 1998) have been identified in plants. These calcium-binding proteins can bind on the order of 20 to 50 calcium ions with both high-(1-3 sites per protein) and low-(20-50 sites per protein) affinity sites. The levels of calcium binding proteins may have a significant impact on signaling processes and may regulate second messenger transmission (Camacho and Lechleiter, 1995;Mery et al., 1996;Coppolino et al., 1997). In an alternative role, calcium-dependent interactions of calnexin and calreticulin have been characterized with a variety of proteins (Nigam et al., 1994;Peterson et al., 1995) and both are implicated in the promotion of correct protein folding (Hebert et al., 1996). These latter activities clearly suggest a molecular chaperone role. Recently, a high-capacity, low-affinity calcium-binding protei...