[Show abstract][Hide abstract] ABSTRACT: Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left-right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.
[Show abstract][Hide abstract] ABSTRACT: A DNA transposon integrated into -the genome of a primitive mammal some 200 million years ago and, millions of years later, it evolved an essential function in the common ancestor of all placental mammals. This protein, now named ZBED6, was recently discovered because a mutation disrupting one of its binding sites, in an intron of the IGF2 gene, makes pigs grow more muscle. These findings have revealed a new mechanism for regulating muscle growth as well as a novel transcription factor that appears to be of major importance for transcriptional regulation in placental mammals.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
The molecular identification of genes and mutations affecting complex traits and disorders has proven to be very challenging in humans as well as in model organisms. These so-called quantitative traits arise from interactions between two or more genes and their environment, and can be mapped to their underlying genes via closely linked stretches of DNA called quantitative trait loci (QTL). Previously, we identified a single nucleotide substitution in a noncoding region of the insulin-like growth factor 2 gene (IGF2) in pigs that is underlying a major QTL affecting muscle growth, heart size, and fat deposition. The mutation disrupts interaction with an unknown nuclear protein acting as a repressor of IGF2 transcription. In the present study, we have isolated a zinc finger protein of unknown function and show that it regulates the expression of IGF2. The protein, which we named ZBED6, is encoded by a domesticated DNA transposon that was inserted into the genome prior to the radiation of placental mammals. ZBED6 is exclusive to placental mammals and highly conserved among species. Our functional characterization of ZBED6 shows that it has a broad tissue distribution and may affect the expression of thousands of other genes, besides IGF2, that control fundamental biological processes. We postulate that ZBED6 is an important transcription factor affecting development, cell proliferation, and growth in placental mammals.
[Show abstract][Hide abstract] ABSTRACT: 5'-AMP-activated protein kinase (AMPK) activity is increased during exercise in an intensity- and glycogen-dependent manner. We previously reported that a mutation in the AMPK3 subunit (Prkag3225Q) increases AMPK activity and skeletal muscle glycogen content. Transfection experiments revealed the R225Q mutation is associated with high basal AMPK activity and diminished AMP dependence. Thus, the R225Q mutation can be considered a loss-of-function mutation that abolished allosteric regulation by AMP/ATP, causing increased basal AMPK activity. We used AMPK3 transgenic (Tg-Prkag3225Q) and knockout (Prkag3-/-) mice to determine the relationship between AMPK activity, glycogen content, and ergogenics (ability to perform work) in isolated extensor digitorum longus skeletal muscle after contractions induced by electrical stimulation. Contraction-induced AMPK activity was inversely coupled to glycogen content in wild-type and Tg-Prkag3225Q mice, but not in Prkag3-/- mice, highlighting a partial feedback control of glycogen on contraction-induced AMPK activity in the presence of a functional AMPK3 isoform. Skeletal muscle glycogen content was positively correlated to work performance, regardless of genotype. Thus, chronic activation of AMPK by the Prkag3225Q mutation directly influences skeletal muscle ergogenics by enhancing glycogen content. In conclusion, functional studies of the AMPK3 isoform further support the close connection between glycogen content and exercise performance in skeletal muscle.
The FASEB Journal 06/2005; 19(7):773-9. DOI:10.1096/fj.04-3221com · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.
[Show abstract][Hide abstract] ABSTRACT: 5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.
[Show abstract][Hide abstract] ABSTRACT: We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low-density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase II and CAMP-/cGMP-dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.
[Show abstract][Hide abstract] ABSTRACT: Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.
[Show abstract][Hide abstract] ABSTRACT: Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.
[Show abstract][Hide abstract] ABSTRACT: The G protein-coupled, extracellular calcium-sensing receptor (CaR) regulates parathyroid hormone secretion and parathyroid cellular proliferation as well as the functions of diverse other cell types. The CaR resides in caveolae-plasma membrane microdomains containing receptors and associated signaling molecules that are thought to serve as cellular "message centers." An additional mechanism for coordinating cellular signaling is the presence of scaffold proteins that bind and organize components of signal transduction cascades. With the use of the yeast two-hybrid system, we identified filamin-A (an actin-cross-linking, putative scaffold protein that binds mitogen-activated protein kinase (MAPK) components activated by the CaR) as an intracellular binding partner of the CaR's carboxyl (COOH)-terminal tail. A direct interaction of the two proteins was confirmed by an in vitro binding assay. Moreover, confocal microscopy combined with two color immunofluorescence showed co-localization of the CaR and filamin-A within parathyroid cells as well as HEK-293 cells stably transfected with the CaR. Deletion mapping localized the sites of interaction between the two proteins to a stretch of 60 amino acid residues within the distal portion of the CaR's COOH-terminal tail and domains 14 and 15 in filamin-A, respectively. Finally, introducing the portion of filamin-A interacting with the CaR into CaR-transfected HEK-293 cells using protein transduction with a His-tagged, Tat-filamin-A fusion protein nearly abolished CaR-mediated activation of ERK1/2 MAPK but had no effect on ERK1/2 activity stimulated by ADP. Therefore, the binding of the CaR's COOH-terminal tail to filamin-A may contribute to its localization in caveolae, link it to the actin-based cytoskeleton, and participate in CaR-mediated activation of MAPK.
[Show abstract][Hide abstract] ABSTRACT: A cDNA of human origin is shown to encode a tRNA isopentenyl transferase (E.C. 184.108.40.206). Expression of the gene in a Saccharomyces cerevisiae mutant lacking the endogenous tRNA isopentenyl transferase MOD5 resulted in functional complementation and reintroduction of isopentenyladenosine into tRNA. The deduced amino acid sequence contains a number of regions conserved in known tRNA isopentenyl transferases. The similarity to the S. cerevisiae MOD5 protein is 53%, and to the Escherichia coli MiaA protein 47%. The human sequence was found to contain a single C2H2 Zn-finger-like motif, which was detected also in the MOD5 protein, and several putative tRNA transferases located by BLAST searches, but not in prokaryotic homologues.
[Show abstract][Hide abstract] ABSTRACT: The release of parathyroid hormone is regulated by the extracellular concentration of Ca2+ through a sensor(s) on the surface of the parathyroid cells, but few details are known on the further relay of the signal inside the cell. Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell soluble fraction to the particulate fraction of the cell. Therefore, identification of a subcellular localization of a PKC isozyme in parathyroid cells as a response to changes in extracellular Ca2+ should be an indication for its putative role in signal transduction coupled to the Ca2+ sensor. We have determined the subcellular localization of six PKC isozymes (alpha, betaI, betaII, epsilon, zeta, and iota) in nonstimulated parathyroid cells and in those treated with low (0.5 mM) and high (3.0 mM) extracellular Ca2+ by confocal microscopy. At the physiological concentration of serum Ca2+, all PKC isozymes studied were localized mainly to the cytosol, although to different extents. Low extracellular Ca2+ caused a redistribution of PKCalpha to the periphery of the cells. In contrast, PKCbetaI, -epsilon, -zeta, and -iota were translocated to the periphery of the cells at high extracellular Ca2+. These results indicate that PKCalpha, -betaI, -epsilon, -zeta, and -iota are involved in the response of parathyroid cells to changes in extracellular Ca2+.
Experimental Cell Research 03/1999; 247(1):9-16. DOI:10.1006/excr.1998.4324 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used riboprobes and monoclonal antibodies to characterize tissue distribution of the human 550-kD homologue to gp330/megalin, primarily identified in the rat kidney. Human gp330/megalin mRNA and protein are readily identified in human parathyroid cells, placental cytotrophoblasts, kidney proximal tubule cells, and epididymal epithelial cells. The immunoreactivity is found on the surface of the cells and is heterogeneously downregulated in parathyroid hyperplasia and adenomas. Cells of the proximal kidney tubule and epididymis express the protein on their luminal aspect. Moreover, the protein is expressed in Type II pneumocytes, mammary epithelial and thyroid follicular cells, and the ciliary body of the eye. Sequence analysis of cDNA fragments, obtained by RT-PCR, revealed identical nucleotide sequences in parathyroid, kidney, placenta, epididymis, and lung. Immunohistochemistry for parathyroid hormone-related protein (PTHrP) revealed partial co-expression with human gp330/megalin in parathyroid, placenta, and mammary gland. The findings substantiate human gp330/megalin expression in a variety of human tissues expected to possess calcium-sensing functions. It may constitute a protein of utmost importance to adult and fetal calcium homeostasis, although other important functions may also be coupled to this exceptionally large protein with highly restricted tissue distribution.
Journal of Histochemistry and Cytochemistry 04/1997; 45(3):383-92. DOI:10.1177/002215549704500306 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase II and cAMP-/cGMP-dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.
European Journal of Biochemistry 08/1996; 239(1):132-7. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, a 500-kDa protein, with homology to the rat Gp330 glycoprotein, was found to be expressed on the surface of human parathyroid, placental cytotrophoblast, and renal proximal tubule cells. The protein has been implicated to function as a sensor of extracellular calcium on parathyroid and placental cytotrophoblast cells. We report here in situ hybridization mapping of the corresponding gene, designed as low-density lipoprotein receptor related protein-2 and symbolized as LRP2, to human chromosome region 2q31-->q32.1 and porcine chromosome region 15q22-->q24. The results are discussed in a comparative mapping context.
Cytogenetics and cell genetics 02/1995; 71(2):120-3. DOI:10.1159/000134088
[Show abstract][Hide abstract] ABSTRACT: Monoclonal anti-parathyroid antibodies have been utilized to isolate a single-chain glycoprotein of 500 kDa, which apparently acts as a sensor of the extracellular calcium concentration and is expressed on the surface of human parathyroid, placental, and kidney tubule cells. The present contribution reports the isolation of a cDNA clone encoding this protein in human placenta and subsequent Northern blots confirming the mRNA expression also in human parathyroid and kidney cells. Close similarity in sequence as well as in tissue distribution is demonstrated with the rat Heymann nephritis antigen, a kidney tubule glycoprotein with calcium-binding ability. The 500-kDa protein belongs to the LDL-receptor superfamily of glycoproteins, claimed to function primarily as protein receptors and characterized by functionally important calcium-binding capacity. It is proposed that the currently identified protein constitutes part of a common structure for the sensing of extracellular calcium concentrations and influences calcium homeostasis in different organs.
Experimental Cell Research 07/1994; 212(2):344-50. DOI:10.1006/excr.1994.1153 · 3.25 Impact Factor