Evolution of the insulin receptor family and receptor isoform expression in vertebrates

The molecular phylogeny of the vertebrate insulin receptor (IR) family was reconstructed under maximum likelihood (ML) to establish homologous relationships among its members. A sister group relationship between the orphan insulin-related receptor (IRR) and the insulin-like growth factor 1 receptor (IGF1R) to the exclusion of the IR obtained maximal bootstrap support. Although both IR and IGF1R were identified in all vertebrates, IRR could not be found in any teleost fish. The ancestral character states at each position of the receptor molecule were inferred for IR, IRR + IGF1R, and all 3 paralogous groups based on the recovered phylogeny using ML in order to determine those residues that could be important for the specific function of IR. For 18 residues, ancestral character state of IR was significantly distinct (probability >0.95) with respect to the corresponding inferred ancestral character states both of IRR + IGF1R and of all 3 vertebrate paralogs. Most of these IR distinct (shared derived) residues were located on the extracellular portion of the receptor (because this portion is larger and the rate of generation of IR shared derived sites is uniform along the receptor), suggesting that functional diversification during the evolutionary history of the family was largely generated modifying ligand affinity rather than signal transduction at the tyrosine kinase domain. In addition, 2 residues at positions 436 and 1095 of the human IR sequence were identified as radical cluster-specific sites in IRR + IGF1R. Both Ir and Irr have an extra exon (namely exon 11) with respect to Igf1r. We used the molecular phylogeny to infer the evolution of this additional exon. The Irr exon 11 can be traced back to amphibians, whereas we show that presence and alternative splicing of Ir exon 11 seems to be restricted exclusively to mammals. The highly divergent sequence of both exons and the reconstructed phylogeny of the vertebrate IR family strongly indicate that both exons were acquired independently by each paralog.     

Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles.

The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post-natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3(-/- )mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation-contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).     

Advancing age alters the expression of the ryanodine receptor 3 isoform in adult rat superior cervical ganglia.

Sympathetic nerves arising from the superior cervical ganglion (SCG) protect the cerebrovasculature during periods of acute hypertension and may play a role in homeostasis of target organs. The functions of these nerves depend on calcium release triggered by activation of ryanodine receptor (RyR) channels. The function of RyR channels is in part dependent on genetic expression and regulation by numerous protein modulators such as neuronal nitric oxide synthase (nNOS) neurons also found in the SCG. We have shown that release of calcium in SCG cells is altered during late maturation and advancing age. However, the underlying molecular mechanisms that may in part account for these data are elusive. Therefore we used molecular techniques to test the hypothesis that advancing age alters the pattern of genetic expression and/or protein levels of RyRs and their modulation by nNOS in the SCG in F344 rats aged 6, 12, and 24 mo. Surprisingly, ryr1 expression was undetectable in all age groups and ryr2 and ryr3 are the predominantly transcribed isoforms in the adult rat SCG. mRNA and protein levels for RyR2 isoform did not change with advancing age. However, ryr3 mRNA levels increased from 6 to 12 mo and declined from 12 to 24 mo. Similarly, RyR3 receptor protein levels also increased from 6 to 12 mo and declined from 12 to 24 mo. Because nNOS and the phosphorylation of the RyRs have been shown to modulate the function of RyRs, total phosphorylation and nNOS protein levels were analyzed in all age groups. Phosphorylation levels of the RyRs were similar in all age groups. However, nNOS protein levels increased from 6 to 12 mo followed by decline from 12 to 24 mo. These data suggest that advancing age selectively impacts the genetic expression and protein levels of RyR3 as well as modulatory nNOS protein levels. In addition, these data may part provide some insight into the possible changes in the function of RyRs that may occur with the normal aging process.     

Properties of ryanodine receptor in rat muscles submitted to unloaded conditions

Unloading of skeletal muscles by hindlimb unweighting is known to induce muscle atrophy and a shift toward faster contractile properties associated with an increase in the expression of fast contractile proteins, particularly in slow soleus muscles. Contractile properties suggest that slow soleus muscles acquire SR properties close to those of a faster one. We studied the expression and properties of the sarcoplasmic reticulum calcium release (RyR) channels in soleus and gastrocnemius muscles of rats submitted to hindlimb unloading (HU). An increase in RyR1 and a slight decrease in RyR3 expression was detected in atrophied soleus muscles only after 4 weeks of HU. No variation appeared in fast muscles. [(3)H]Ryanodine binding experiments showed that HU neither increased the affinity of the receptors for [(3)H]ryanodine nor changed the caffeine sensitivity of [(3)H]ryanodine binding. Our results suggested that not only RyR1 but also RyR3 expression can be regulated by muscle activity and innervation in soleus muscle. The changes in the RyR expression in slow fibers suggested a transformation of the SR from a slow to a fast phenotype.     

Dantrolene inhibition of ryanodine receptor Ca2+ release channels. Molecular mechanism and isoform selectivity

As an inhibitor of Ca(2+) release through ryanodine receptor (RYR) channels, the skeletal muscle relaxant dantrolene has proven to be both a valuable experimental probe of intracellular Ca(2+) signaling and a lifesaving treatment for the pharmacogenetic disorder malignant hyperthermia. However, the molecular basis and specificity of the actions of dantrolene on RYR channels have remained in question. Here we utilize [(3)H]ryanodine binding to further investigate the actions of dantrolene on the three mammalian RYR isoforms. The inhibition of the pig skeletal muscle RYR1 by dantrolene (10 microm) was associated with a 3-fold increase in the K(d) of [(3)H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles such that dantrolene effectively reversed the 3-fold decrease in the K(d) for [(3)H]ryanodine binding resulting from the malignant hyperthermia RYR1 Arg(615) --> Cys mutation. Dantrolene inhibition of the RYR1 was dependent on the presence of the adenine nucleotide and calmodulin and reflected a selective decrease in the apparent affinity of RYR1 activation sites for Ca(2+) relative to Mg(2+). In contrast to the RYR1 isoform, the cardiac RYR2 isoform was unaffected by dantrolene, both in native cardiac SR vesicles and when heterologously expressed in HEK-293 cells. By comparison, the RYR3 isoform expressed in HEK-293 cells was significantly inhibited by dantrolene, and the extent of RYR3 inhibition was similar to that displayed by the RYR1 in native SR vesicles. Our results thus indicate that both the RYR1 and the RYR3, but not the RYR2, may be targets for dantrolene inhibition in vivo.     

Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor

Expression of the inhibitory glycine receptor complex was investigated in primary cultures of fetal mouse spinal cord using sensitive immunomethods. In these cells, glycine receptor is predominantly of the neonatal isoform characterized by a low affinity for the antagonist strychnine. It contains a ligand binding subunit that differs from that of the adult receptor in antigenic epitopes and apparent molecular weight. Whereas in vivo the neonatal receptor isoform is completely replaced by the adult isoform within 3 weeks after birth, this exchange of subtypes is not seen in culture. The increased expression of the cytoplasmic glycine receptor-associated polypeptide of 93 kd occurring after birth is also seen under culture conditions. Purification of glycine receptor from cultures yielded polypeptides of 49 kd and 93 kd, suggesting that the membrane-spanning core of the neonatal receptor may be a homooligomer composed of 49 kd subunits. About half of the 49 kd subunit is cleaved by trypsinization of the cultures, indicating a predominant cell surface localization of the receptor. Pulse-labeling experiments revealed the 49 kd subunit to be a metabolically stable glycoprotein (half-life approximately 2 days). After its synthesis, a transition time of 30-45 min is required for acquisition of a strychnine binding conformation.     

Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family.

We have isolated and sequenced a full-length cDNA clone encoding rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C.1.2.1.12). The entire mRNA is 1269 nucleotides long exclusive of poly(A) and contains respectively 71 and 196 bases of 5' and 3' non-coding regions. Primer extension as well as S1 nuclease protection experiments clearly established that a single (or at least a highly prominent) GAPDH mRNA species is expressed in all rat tissues examined. This sequence allowed the determination of the hitherto unknown primary structure of rat GAPDH which is 333 aminoacids long. Comparison between GAPDH sequences from rat, man and chicken revealed a high degree of sequence conservation at both nucleotide and protein levels.     

Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1

The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.     

The Drosophila Activin-like ligand Dawdle signals preferentially through one isoform of the Type-I receptor Baboon

How TGF-β-type ligands achieve signaling specificity during development is only partially understood. Here, we show that Dawdle, one of four Activin-type ligands in Drosophila, preferentially signals through Babo_c, one of three isoforms of the Activin Type-I receptor that are expressed during development. In cell culture, Dawdle signaling is active in the presence of the Type-II receptor Punt but not Wit, demonstrating that the Type-II receptor also contributes to the specificity of the signaling complex. During development, different larval tissues express unique combinations of these receptors, and ectopic expression of Babo_c in a tissue where it is not normally expressed at high levels can make that tissue sensitive to Dawdle signaling. These results reveal a mechanism by which distinct cell types can discriminate between different Activin-type signals during development as a result of differential expression of Type-I receptor isoforms.     

Characterization of a novel and functional human prolactin receptor isoform (deltaS1PRLr) containing only one extracellular fibronectin-like domain

Prolactin (PRL)-dependent signaling occurs as the result of ligand-induced homodimerization of the PRL receptor (PRLr). To date, short, intermediate, and long human PRLr isoforms have been characterized. To investigate the expression of other possible human PRLr isoforms, RT-PCR was performed on mRNA isolated from the breast carcinoma cell line T47D. A 1.5-kb PCR fragment was isolated, subcloned, and sequenced. The PCR product exhibited a nucleotide sequence 100% homologous to the human long isoform except bp 71-373 were deleted, which code for the S1 motif of the extracellular domain. Therefore, this isoform was designated the deltaS1 PRLr. Northern analysis revealed variable deltaS1 PRLr mRNA expression in a variety of tissues. Transfection of Chinese hamster ovary cells with deltaS1 cDNA showed the isoform is expressed at the protein level on the cell surface with a molecular mass of approximately 70 kDa. Kinetic studies indicated the deltaS1 isoform bound ligand at a lower affinity than wild-type receptor. The deltaS1 PRLr was also shown to activate the proximal signaling molecule Jak2 upon addition of ligand to transfected cells, and, unlike the long PRLr, high concentrations of ligand did not function as a self-antagonist to signaling during intervals of PRL serum elevation, i.e. stress and pregnancy. Given its apparent widespread expression, this PRLr isoform may contribute to PRL action. Furthermore, the functionality of this receptor raises interesting questions regarding the minimal extracellular domain necessary for ligand-induced receptor signaling.     

The interaction of spermine with the ryanodine receptor from skeletal muscle.

The effect of polyamines on ryanodine binding activity of junctional sarcoplasmic reticulum membranes is described. Spermine stimulated the binding of ryanodine to its receptor up to 5-fold, with half-maximal stimulation obtained with 3.5 mM. Spermidine and putrescine also stimulated ryanodine binding, but they were about 12-fold less potent. The degree of stimulation is dependent on the NaCl concentration present in the assay medium. Spermine has no effect on the Ca(2+)-dependency of ryanodine binding but it increases the ryanodine binding affinity (Kd) by about 5.6-fold. Both the rate of ryanodine association with, and dissociation from, its binding site were affected by spermine. Spermine also stimulates the photoaffinity labelling by 3-O-(4-benzoyl)benzoyl[alpha-32P]ATP ([alpha-32P]BzATP) of the ryanodine receptor, increasing the BzATP binding affinity. We suggest that the stimulatory effect of spermine on ryanodine binding is due to its specific interaction with the ryanodine receptor. This spermine interaction enabled us to develop a new, one-step, fast and with high yield method for the purification of ryanodine receptor (Shoshan-Barmatz, V. and Zarka, A. (1992) Biochem. J. 284, in press).     

Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish

We have cloned agroup of cDNAs that encodes the skeletal ryanodine receptor isoform(RyR1) of fish from a blue marlin extraocular muscle library. The cDNAsencode a protein of 5,081 amino acids with a calculated molecular massof 576,302 Da. The deduced amino acid sequence shows strong sequenceidentity to previously characterized RyR1 isoforms. An RNA probederived from a clone of the full-length marlin RyR1 isoform hybridizesto RNA preparations from extraocular muscle and slow-twitch skeletalmuscle but not to RNA preparations from fast-twitch skeletal or cardiacmuscle. We have also isolated a partial RyR clone from marlin andtoadfish fast-twitch muscles that shares 80% sequence identity withthe corresponding region of the full-length RyR1 isoform, and a RNAprobe derived from this clone hybridizes to RNA preparations fromfast-twitch muscle but not to slow-twitch muscle preparations. Westernblot analysis of slow-twitch muscles in fish indicates the presence ofonly a single high-molecular-mass RyR proteincorresponding to RyR1. [³H]ryanodine bindingassays revealed the fish slow-twitch muscle RyR1 had a greatersensitivity for Ca²⁺ than thefast-twitch muscle RyR1. The results indicate that, in fish muscle,fiber type-specific RyR1 isoforms are expressed and the two proteinsare physiologically distinct.

     

Studies of the diffuse x-ray scattering from contracting frog skeletal muscles.

Using x-rays from synchrotron radiation, we studied diffuse scattering, sometimes together with the myosin layer lines. With an area detector, sartorius muscles and a time resolution of 150 ms, earlier results from semitendinosus muscles contracting isometrically at 6 degrees C (Lowy, J., and F. R. Poulsen. 1987. J. Mol. Biol. 194:595-600) were confirmed and extended. Evidence from intensity changes both in the diffuse scattering and in the myosin layer lines showed that the majority of the heads become disordered at peak tetanic tension. With a linear detector and a time resolution of 5 ms, it was found that during tension rise the intensity increase of the diffuse scattering (which amounted maximally to 12% recorded near the meridian) runs approximately 20 ms ahead of the mechanical change, comparing half-completion times. This suggests that an appreciable number of heads change orientation before peak tension is reached. In quick release experiments the diffuse scattering intensity showed very little change. Recorded near the meridian during rapid shortening, however, it decreased progressively with a half-time of approximately 40 ms. This change amounted to approximately 35% of that observed during the initial tension rise. We interpret this to indicate that during rapid shortening a certain number of heads assume an orientation characteristic of the relaxed state. Viewed in the context of the behavior of the first myosin layer line and the (1, 1) equatorial reflection in similar experiments (Huxley, H. E., M. Kress, A. R. Faruqi, and R. M. Simmons. 1988.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy

Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly.     

An insertion within a variably spliced Drosophila tropomyosin gene blocks accumulation of only one encoded isoform

We have characterized an aberrant allele of a variably spliced Drosophila tropomyosin gene. The allele was recovered from the flightless Ifm(3)3 strain, which has been shown to have structurally and functionally abnormal indirect flight muscles. The transcribed portion of the mutant gene is interrupted by an 8,8 kb insertion of middle repetitive DNA having a structure typical of copia-like Drosophila mobile elements. The insertion is positioned so as to interrupt an exon sequence in one splicing mode and, simultaneously, an intron in the alternate mode. As a consequence of the insertion the allele fails to direct synthesis of the flight muscle-specific tropomyosin isoform, but remains capable of specifying a second isoform, which accumulates in nonfibrillar Drosophila muscles.