Ageing, but not yet senescent, rats exhibit reduced muscle quality and sarcoplasmic reticulum function.

AIM: Reduced muscle force greater than expected from loss of muscle mass has been reported in ageing muscles. Impaired sarcoplasmic reticulum (SR) Ca(2+) release has been implicated as a possible mechanism, and attributed to several factors, including loss of ryanodine receptor (RYR) expression and protein binding. The aim of this study was to evaluate muscle quality and SR Ca(2+) release in ageing rats that were not so old that major atrophy had occurred. METHODS: We collected in situ force data from the plantarflexor muscle group and muscle mass from the constituent muscles to determine muscle quality (force/mass) in adult (6-8 months) and ageing (24 months) rats (n=8/group). We evaluated SR Ca(2+) uptake and release, and determined expression of key proteins associated with Ca(2+) release [RYR and FK506 binding protein (FKBP)] and uptake (SERCA, parvalbumin, calsequestrin). RESULTS: Plantarflexor force and muscle quality were reduced with ageing (approx. 28 and 34%, respectively), but atrophy was limited, and significant only in the medial gastrocnemius (approx. 15%). The fast phase of SR Ca(2+) release was reduced with ageing in both gastrocnemii, as was FKBP expression and FKBP-RYR binding, but RYR expression was not affected. Similar, but non-significant changes were present in the plantaris, but the soleus muscle generally showed no ageing-related changes. CONCLUSION: These data suggest a possible role for impaired SR Ca(2+) release in ageing-related loss of muscle quality, although not through loss of RYR expression.

Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats.

Defective calcium (Ca2+) signaling and impaired contractile function have been observed in skeletal muscle secondary to impaired myocardial function. However, the molecular basis for these muscle defects have not been identified. In this study, we evaluated the alterations of the ryanodine-sensitive Ca2+ release channels (RyR1) by analyzing global and local Ca2+ signaling in a rat postmyocardial infarction (PMI) model of myocardial overload. Ca2+ transients, measured with multiphoton imaging in individual fibers within a whole extensor digitorum longus (EDL) muscle, exhibited significantly reduced amplitude and a prolonged time course in PMI. Spatio-temporal properties of spontaneous Ca2+ sparks in fibers isolated from PMI EDL muscles were also significantly altered. In addition, RyR1 from PMI skeletal muscles were PKA-hyperphosphorylated and depleted of the FK506 binding protein (FKBP12). These data show that PMI skeletal muscles exhibit altered local Ca2+ signaling, associated with hyperphosphorylation of RyR1. The observed changes in Ca2+ signaling may contribute to defective excitation-contraction coupling in muscle that can contribute to the reduced exercise capacity in PMI, out of proportion to the degree of cardiac dysfunction.

Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system.

The relationship between the L1 and L2 domains of the insulin and epidermal growth factor receptors and leucine-rich repeat modules.

BACKGROUND: Leucine-rich repeats are one of the more common modules found in proteins. The leucine-rich repeat consensus motif is LxxLxLxxNxLxxLxxLxxLxx- where the first 11-12 residues are highly conserved and the remainder of the repeat can vary in size Leucine-rich repeat proteins have been subdivided into seven subfamilies, none of which include members of the epidermal growth factor receptor or insulin receptor families despite the similarity between the 3D structure of the L domains of the type I insulin-like growth factor receptor and some leucine-rich repeat proteins. RESULTS: Here we have used profile searches and multiple sequence alignments to identify the repeat motif Ixx-LxIxx-Nx-Lxx-Lxx-Lxx-Lxx- in the L1 and L2 domains of the insulin receptor and epidermal growth factor receptors. These analyses were aided by reference to the known three dimensional structures of the insulin-like growth factor type I receptor L domains and two members of the leucine rich repeat family, porcine ribonuclease inhibitor and internalin 1B. Pectate lyase, another beta helix protein, can also be seen to contain the sequence motif and much of the structural features characteristic of leucine-rich repeat proteins, despite the existence of major insertions in some of its repeats. CONCLUSION: Multiple sequence alignments and comparisons of the 3D structures has shown that right-handed beta helix proteins such as pectate lyase and the L domains of members of the insulin receptor and epidermal growth factor receptor families, are members of the leucine-rich repeat superfamily.

Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain.

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.

The three dimensional structure of the type I insulin-like growth factor receptor.

Ever since the discovery of insulin and its role in the regulation of glucose metabolism, there has been great interest in the molecule itself, the insulin-like growth factors (IGFs), and their receptors (IR and IGF-R). These receptors form a subfamily of tyrosine kinase receptors which are large, transmembrane proteins consisting of several structural domains. Their ectodomains have a similar arrangement of two homologous domains (L1 and L2) separated by a Cys rich region. The C-terminal half of their ectodomains consists of three fibronectin type 3 repeats, and an insert domain that contains the alpha-beta cleavage site. This review summarises the key developments in the understanding of the structure of this family of receptors and their relation to other multidomain proteins. Data presented will include multiple sequence analyses, single molecule electron microscope images of the IGF-1R, insulin receptor (IR), and IR-Fab complexes, and the three dimensional structure of the first three domains of the IGF-1R determined to 2.6 A resolution by x ray crystallography. The L domains each adopt a compact shape consisting of a single stranded, right handed beta-helix. The Cys rich region is composed of eight disulphide bonded modules, seven of which form a rod shaped domain with modules associated in an unusual manner.

Definition of a minimal munc18c domain that interacts with syntaxin 4.

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein-protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225), but not munc18c(226-592), munc18c(1-100), munc18c(43-139) or munc18c(66-139), interacted with the cytoplasmic portion of syntaxin 4, Stx4(2-273), as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by beta-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx4(29-157), failed to interact with full-length munc18c(1-592), indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein-protein interaction studies between Stx4(2-273) and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225) interacted with Stx4(2-273) whereas munc18c(1-100) did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

Structure and function of the type 1 insulin-like growth factor receptor.

The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.

Effects of imperatoxin A on local sarcoplasmic reticulum Ca(2+) release in frog skeletal muscle.

We have investigated the effects of imperatoxin A (IpTx(a)) on local calcium release events in permeabilized frog skeletal muscle fibers, using laser scanning confocal microscopy in linescan mode. IpTx(a) induced the appearance of Ca(2+) release events from the sarcoplasmic reticulum that are approximately 2 s and have a smaller amplitude (31 +/- 2%) than the "Ca(2+) sparks" normally seen in the absence of toxin. The frequency of occurrence of long-duration imperatoxin-induced Ca(2+) release events increased in proportion to IpTx(a) concentrations ranging from 10 nM to 50 nM. The mean duration of imperatoxin-induced events in muscle fibers was independent of toxin concentration and agreed closely with the channel open time in experiments on isolated frog ryanodine receptors (RyRs) reconstituted in planar lipid bilayer, where IpTx(a) induced opening of single Ca(2+) release channels to prolonged subconductance states. These results suggest involvement of a single molecule of IpTx(a) in the activation of a single Ca(2+) release channel to produce a long-duration event. Assuming the ratio of full conductance to subconductance to be the same in the fibers as in bilayer, the amplitude of a spark relative to the long event indicates involvement of at most four RyR Ca(2+) release channels in the production of short-duration Ca(2+) sparks.

High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper.

Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors. These soluble IRs have lowered affinity for insulin compared with full-length IR, and exhibit linear Scatchard plots in contrast to the curvilinear plots obtained with full-length IR, IR truncated at the C-terminus of the transmembrane region and IR ectodomains fused to the self-associating constant domains from Fc or lambda immunoglobulins. In this report, we have fused the IR ectodomain to the 33 residue leucine zipper from the transcriptional activator GCN4 of Saccharomyces cerevisiae. This fusion protein binds insulin with high affinity in a manner comparable to native receptor. The respective dissociation constants were Kd1 8.2 X 10(-11) M and Kd2 1.6 x 10(-8) M for hIRedZip and Kd1 5.7 x 10(-11) M and Kd2 6.3 x 10(-9) M for membrane-anchored, native receptor.

Two mechanisms for termination of individual Ca2+ sparks in skeletal muscle.

Ca(2+) sparks are brief, localized elevations of myoplasmic [Ca(2+)] caused by release of increments of Ca(2+) via sarcoplasmic reticulum Ca(2+) release channels in muscle. The properties of individual sparks provide information regarding the opening of sarcoplasmic reticulum Ca(2+) channels within functioning cells. Here we use high-speed confocal microscopy to show that individual Ca(2+) sparks activated by membrane depolarization in single frog skeletal muscle fibers can be terminated prematurely by repolarization. Thus, either voltage sensor deactivation on repolarization or release channel inactivation during continued depolarization can terminate the Ca(2+) release channel activity underlying voltage-activated Ca(2+) sparks in skeletal muscle.

Expression of ryanodine receptor RyR3 produces Ca2+ sparks in dyspedic myotubes.

Discrete, localized elevations of myoplasmic [Ca2+], Ca2+ 'sparks', were readily detected using the fluorescent Ca2+ indicator fluo-3 and laser scanning confocal microscopy in 'dyspedic' 1B5 myotubes, i.e. myotubes which do not express ryanodine receptors (RyRs), transduced with virions containing cDNA for RyR type 3 that were saponin permeabilized to allow dye entry. Ca2+ sparks were never observed in non-transduced RyR null myotubes. The spatial locations of sparks observed in permeabilized myotubes roughly corresponded to regions of RyR protein expression in the same myotube as detected after subsequent fixation and antibody staining. Permeabilized RyR3-transduced myotubes exhibited similar punctate peripheral RyR3 protein immunohistochemical patterns as myotubes fixed before permeabilization indicating that permeabilization did not affect the structural organization of the triad. Ca2+ sparks, recorded in line scan mode, in permeabilized myotubes expressing RyR3 exhibited mean amplitudes (change in fluorescence/mean fluorescence, DeltaF/F: 1.20 +/- 0.04) and temporal rise times (10-90%; 6.31 +/- 0.12 ms) similar to those of sparks recorded in permeabilized frog skeletal muscle fibres (0.98 +/- 0.01; 6.11 +/- 0.07, respectively) using the same confocal system. Spatial extent and temporal duration of the Ca2+ sparks were approximately 40% larger in the RyR3-expressing myotube cultures than in frog fibres. Ca2+ sparks recorded in line scan mode often occurred repetitively at the same spatial location in RyR3-expressing myotubes. Such repetitive events were highly reproducible in amplitude and spatio-temporal properties, as previously observed for repetitive mode sparks in frog skeletal muscle. Ca2+ sparks recorded in xy mode were frequently compressed in the y (slower scan) direction compared to the x direction. This asymmetry was reproduced assuming spatially symmetric events having the time course of Ca2+ sparks recorded in line scan (xt) mode. These expression studies demonstrate that the presence of RyR3 is sufficient for the production of Ca2+ sparks in a skeletal muscle system lacking the expression of any other RyR isoform.

Mutational analysis of the N-linked glycosylation sites of the human insulin receptor.

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into alpha and beta subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (Delta6) and 16+295+337+418+730+743+881 (Delta7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (Delta7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of Delta7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of Delta6 and Delta7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The Delta6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.

Properties of an insulin receptor with an IGF-1 receptor loop exchange in the cysteine-rich region.

The insulin receptor (IR) and the insulin-like growth factor-I receptor (IGF-1R) show differential binding of insulin and IGFs. The specificity determinants for IGF-1 binding are known to be located in the cysteine-rich (Cys-rich) region between residues 223 and 274 of human IGF-1R, which includes a loop that protrudes into the putative ligand binding site. In this report we have replaced residues 260-277 of human IR with residues 253-266 of the human IGF-1R to produce an IR-based, cysteine loop exchange chimaera, termed hIR-Cys loop exchange (CLX), in which all 14 amino acid residues in the exchanged loop differ from wild-type insulin receptor. This loop exchange had a detrimental effect on the efficiency of pro-receptor processing and on the binding of the mouse monoclonal antibody 83-7. However, this antibody, which binds hIR but not hIGF-1R, was still capable of immunoprecipitating the mature chimaeric receptor, indicating that the conformational epitope recognised by this antibody is not primarily determined by the loop region exchanged. The loop exchange did not significantly affect the ability of insulin to displace bound radiolabelled insulin, but increased the capacity of IGF-1 to competitively displace labelled insulin by at least 10 fold.

Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor.

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.

Members of the insulin receptor family contain three fibronectin type III domains.

The insulin receptor is a large transmembrane dimer, comprised of several domains. Detailed 3D structural information is available for the L1-cys-rich-L2 domains in the extracellular region (ectodomain) and for the tyrosine kinase catalytic domain in the cytoplasmic portion of the receptor. In addition, previous sequence analyses have identified two fibronectin type III domains in the C-terminal half of each ectodomain monomer. In this report, evidence is provided to show that a third fibronectin type III module exists between the L2 domain and the two previously described fibronectin type III domains.

Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.

The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.

Single-molecule imaging of human insulin receptor ectodomain and its Fab complexes.

The insulin receptor (IR) is a four-chain, transmembrane dimer held together by disulfide bonds. To gain information about the molecular envelope and the organization of its domains, single-molecule images of the IR ectodomain and its complexes with three Fabs have been analyzed by electron microscopy. The data indicate that the IR ectodomain resembles a U-shaped prism of approximate dimensions 90 x 80 x 120 A. The width of the cleft (assumed membrane-distal) between the two side arms is sufficient to accommodate ligand. Fab 83-7, which recognizes the cys-rich region of IR, bound halfway up one end of each side arm in a diametrically opposite manner, indicating a twofold axis of symmetry normal to the membrane surface. Fabs 83-14 and 18-44, which have been mapped respectively to the first fibronectin type III domain (residues 469-592) and residues 765-770 in the insert domain, bound near the base of the prism at opposite corners. These images, together with the data from the recently determined 3D structure of the first three domains of the insulin-like growth factor type I receptor, suggest that the IR dimer is organized into two layers with the L1/cys-rich/L2 domains occupying the upper (membrane distal) region of the U-shaped prism and the fibronectin type III domains and the insert domains located predominantly in the membrane-proximal region.

Time course of individual Ca2+ sparks in frog skeletal muscle recorded at high time resolution.

Discrete Ca2+ release events (Ca2+ "sparks") were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 microseconds per line). Fibers loaded with the Ca2+ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at -90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca2+ sparks at approximately 30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5-2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 +/- 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca2+ ions are being released by the sarcoplasmic reticulum Ca2+ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca2+ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.

Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor.

The type-1 insulin-like growth-factor receptor (IGF-1R) and insulin receptor (IR) are closely related members of the tyrosine-kinase receptor superfamily. IR is essential for glucose homeostasis, whereas IGF-1R is involved in both normal growth and development and malignant transformation. Homologues of these receptors are found in animals as simple as cnidarians. The epidermal growth-factor receptor (EGFR) family is closely related to the IR family and has significant sequence identity to the extracellular portion we describe here. We now present the structure of the first three domains of IGF-IR (L1-Cys-rich-L2) determined to 2.6 A resolution. The L domains each consist of a single-stranded right-handed beta-helix. The Cys-rich region is composed of eight disulphide-bonded modules, seven of which form a rod-shaped domain with modules associated in an unusual manner. The three domains surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment (residues 1-462) does not bind ligand, many of the determinants responsible for hormone binding and ligand specificity map to this central site. This structure therefore shows how the IR subfamily might interact with their ligands.