Terminal differentiation of keratinocytes involves the sequential expression of several major proteins which can be identified in distinct cellular layers within the mammalian epidermis and are characteristic for the maturation state of the keratinocyte. Many of the corresponding genes are clustered in one specific human chromosomal region 1q21. It is rare in the genome to find in such close proximity the genes belonging to at least three structurally different families, yet sharing spatial and temporal expression specificity, as well as interdependent functional features. This DNA segment, termed the epidermal differentiation complex, contains 27 genes, 14 of which are specifically expressed during calcium-dependent terminal differentiation of keratinocytes (the majority being structural protein precursors of the cornified envelope) and the other 13 belong to the S100 family of calcium binding proteins with possible signal transduction roles in the differentiation of epidermis and other tissues. In order to provide a bacterial clone resource that will enable further studies of genomic structure, transcriptional regulation, function and evolution of the epidermal differentiation complex, as well as the identification of novel genes, we have constructed a single 2.45 Mbp long continuum of genomic DNA cloned as 45 p1 artificial chromosomes, three bacterial artificial chromosomes, and 34 cosmid clones. The map encompasses all of the 27 genes so far assigned to the epidermal differentiation complex, and integrates the physical localization of these genes at a high resolution on a complete NotI and SalI, and a partial EcoRI restriction map. This map will be the starting resource for the large-scale genomic sequencing of this region by The Sanger Center, Hinxton, U.K.

The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, ؊TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse.The ataxic and epileptic mutant mouse, stargazer (stg), arose spontaneously as a consequence of a viral insertion of a 6-kb early transposon in intron 2 of the stargazin gene (1, 2). The mutation results in premature transcriptional arrest and complete ablation of stargazin expression (3, 4). From P14 onwards stg display phenotypic consequences of the mutation that includes head tossing due to an inner ear defect (2), ataxia and impaired conditioned eyeblink reflex, both a consequence of cerebellar defects (5) and absence epilepsy (6). The molecular basis for these disparate defects has still to be unequivocally resolved but ultimately these must be direct or downstream consequences of ablated expression of stargazin. Based on low sequence homology to the skeletal muscle-specific L-type voltage-gated calcium channel (VGCC) 1 ␥ 1 subunit, stargazin was proposed to be a brain-specific ␥ isoform, and in this context was named CACN␥ 2 (2). Heterologous co-expression studies showed that stargazin had relatively minor effects on P/Q-, Land ␣1L T-type VGCC kinetics and cell surface trafficking (2, 7, 8, 9, 10). It has recently been shown that the N-type VGCC ␣1B subunit co-precipitates with immunoprecipitated stargazin from detergent soluble mouse brain (4). Cerebellar GABA A receptor expression is also severely compromised in stargazer mice (11,12). The apparent f...

In view of its central role in glycolysis and gluconeogenesis and its polymorphic genetic variability, the phosphoglucomutase 1 (PGM1) gene in man has been the target of protein structural studies and genetic analysis for more than 25 years. We have now isolated genomic clones containing the complete PGM1 gene and have shown that it spans over 65 kb and contains 11 exons. We have also shown that the sites of the two mutations which form the molecular basis for the common PGM1 protein polymorphism lie in exons 4 and 8 and are 18 kb apart. Within this region there is a site of intragenic recombination. We have discovered two alternatively spliced first exons, one of which, exon 1A, is transcribed in a wide variety of cell types; the other, exon 1B, is transcribed in fast muscle. Exon 1A is transcribed from a promoter which has the structural hallmarks of a housekeeping promoter but lies more than 35 kb upstream of exon 2. Exon 1B lies 6 kb upstream of exon 2 within the large first intron of the ubiquitously expressed PGM1 transcript. The fast-muscle form of PGM1 is characterized by 18 extra amino acid residues at its N-terminal end. Sequence comparisons show that exons 1A and 1B are structurally related and have arisen by duplication.

Stargazer (stg) mutant mice fail to express stargazin [transmembrane AMPA receptor regulatory protein ␥2 (TARP␥2)] and consequently experience absence seizure-like thalamocortical spike-wave discharges that pervade the hippocampal formation via the dentate gyrus (DG). As in other seizure models, the dentate granule cells of stg develop elaborate reentrant axon collaterals and transiently overexpress brain-derived neurotrophic factor. We investigated whether GABAergic parameters were affected by the stg mutation in this brain region. GABA A receptor (GABAR) ␣4 and ␤3 subunits were consistently upregulated, GABAR ␦ expression appeared to be variably reduced, whereas GABAR ␣1, ␤2, and ␥2 subunits and the GABAR synaptic anchoring protein gephyrin were essentially unaffected. We established that the ␣4␤␥2 subunit-containing, flunitrazepam-insensitive subtype of GABARs, not normally a significant GABAR in DG neurons, was strongly upregulated in stg DG, apparently arising at the expense of extrasynaptic ␣4␤␦-containing receptors. This change was associated with a reduction in neurosteroid-sensitive GABAR-mediated tonic current. This switch in GABAR subtypes was not reciprocated in the tottering mouse model of absence epilepsy implicating a unique, intrinsic adaptation of GABAergic networks in stg.Contrary to previous reports that suggested that TARP␥2 is expressed in the dentate, we find that TARP␥2 was neither detected in stg nor control DG. We report that TARP␥8 is the principal TARP isoform found in the DG and that its expression is compromised by the stargazer mutation. These effects on GABAergic parameters and TARP␥8 expression are likely to arise as a consequence of failed expression of TARP␥2 elsewhere in the brain, resulting in hyperexcitable inputs to the dentate.

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Stargazer mice fail to express the ␥2 isoform of transmembrane ␣-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 ؎ 6% fewer inhibitory ␥-aminobutyric acid, type A (GABA A ), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABA A receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (␣6␤x␦) ␣6-containing GABA A receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABA A receptor ␣6, ␦ (mature cerebellar granule cell-specific proteins), and ␤3 subunit expression in stargazer. The subunit composition of the residual ␣6-containing GABA A receptors was unaffected by the stargazer mutation. However, we did find evidence of an ϳ4-fold up-regulation of ␣1␤␦ receptors that may compensate for the loss of ␣6-containing GABA A receptors. PCR analysis identified a dramatic reduction in the steady-state level of ␣6 mRNA, compatible with ␣6 being the primary target of the stargazer mutation-mediated GABA A receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic ␣6-containing GABA A receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.

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