Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy.

Although cytoskeletal mutations are known causes of genetically based forms of dilated cardiomyopathy, the pathways that link these defects with cardiomyopathy are unclear. Here we report that the alpha-actinin-associated LIM protein (ALP; Alp in mice) has an essential role in the embryonic development of the right ventricular (RV) chamber during its exposure to high biomechanical workloads in utero. Disruption of the gene encoding Alp (Alp) is associated with RV chamber dilation and dysfunction, directly implicating alpha-actinin-associated proteins in the onset of cardiomyopathy. In vitro assays showed that Alp directly enhances the capacity of alpha-actinin to cross-link actin filaments, indicating that the loss of Alp function contributes to destabilization of actin anchorage sites in cardiac muscle. Alp also colocalizes at the intercalated disc with alpha-actinin and gamma-catenin, the latter being a known disease gene for human RV dysplasia. Taken together, these studies point to a novel developmental pathway for RV dilated cardiomyopathy via instability of alpha-actinin complexes.

Structure and expression of the insulin-like peptide receptor from amphioxus.

Insulin and the insulin-like growth factors IGF-1 and IGF-II are found in all vertebrates, and these anabolic peptides share primary and tertiary structural features which suggest that they have evolved from a common ancestral gene. We have proposed that an insulin-like peptide (ILP) cDNA recently cloned from the protochodate amphioxus may represent the ancestral gene in that the deduced sequence of ILP contains features of both insulin and IGF, and it evidently represents a hybrid insulin/IGF molecule. To expand this hypothesis we have cloned the cDNA that encodes the cognate receptor from amphioxus. Primary sequence comparisons show that the ILP receptor is a member of the insulin receptor family, which in mammals includes the insulin receptor (IR), type I IGF receptor (IGF-IR), and IR-related receptor (IRR). In overall amino acid sequence, the ILP receptor is 48.6% identical to the human (h)IR, 47.3% identical to hIGF-IR, and 43.7% identical to hIRR, and this contrasts with the finding that hIR and hIGF-IR share 57.6% identity. Using degenerate oligonucleotide primers, we show by RT-PCR that amphioxus contains only a single member of the insulin receptor gene family. To complement the sequence comparison, we expressed the ILP receptor protein by transfecting the cDNA into 293 cells. Autophosphorylation of the expressed ILP receptor was half-maximally stimulated by a synthetic ILP analog, (B1-Thr)ILP, at a concentration of about 5 x 10(-7) M. Interestingly, autophosphorylation of the ILP receptor was also stimulated by incubation with either mammalian insulin or IGF-I, although equally high concentrations (10(-5) M) of each were required. Based on these results, we propose that, analogously to the ILP gene, the ancestral ILP receptor gene also duplicated and diverged to generate the IR and IGF-IR genes during the evolutionary transition from protochordates to vertebrates. Our results also indicate that the amphioxus ILP receptor contains the basic structural determinants that are necessary for binding and activation by mammalian insulin and IGF-I.

Genetic basis of hypoxanthine guanine phosphoribosyltransferase deficiency in a patient with the Lesch-Nyhan syndrome (HPRTFlint).

The molecular basis for complete hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency has been determined in a patient with Lesch-Nyhan syndrome. A B-lymphoblastoid cell line derived from this patient expresses normal amounts of HPRT mRNA yet no detectable immunoreactive protein as determined by radioimmunoassay. These findings suggest either a decreased rate of translation or accelerated degradation due to enhanced proteolytic susceptibility. cDNAs synthesized from this patient's RNA have a single nucleotide (nt) substitution, a C----A transversion at nt 222. RNase A cleavage analysis confirms the presence of a mutation at this position within mRNA isolated from lymphoblasts from patient A.C. This transversion predicts a phenylalanine to leucine replacement at amino acid position 73 in the translated protein. We have designated this mutant HPRTFlint. The mutation in HPRTFlint disrupts a strongly conserved region among PRTases from Escherichia coli, rodents and man, suggesting an important role for this region for the normal function of HPRT. Since it is unlikely that this amino acid substitution alters the translational rate, we hypothesize that disruption of the secondary structure within this region renders HPRTFlint more susceptible to proteolysis.

Fusion of insulin receptor ectodomains to immunoglobulin constant domains reproduces high-affinity insulin binding in vitro.

A unique feature of the insulin receptor is that it is dimeric in the absence of ligand. Dimerization of two adjacent transmembrane domain (TMD) alpha helices has been shown to be critical in receptor kinase activation. Moreover, previous work has suggested that the TMD is involved in stabilizing the high-affinity binding site; soluble receptors expressed after simple truncation at the ectodomain-TMD junction have reduced affinity for insulin. To further examine this issue, we have replaced the TMD and intracellular domain of the soluble human insulin receptor (HIRs) with constant domains from immunoglobulin Fc and lambda subunits (HIRs-Fc and HIRs-lambda). Studies of receptor biosynthesis and binding characteristics were performed following transient transfection of receptor cDNAs into human embryonal kidney 293 cells. Each hybrid receptor was initially synthesized as a single chain proreceptor, followed by cleavage into alpha- and beta-Fc or beta-lambda subunits. The majority of secreted protein appeared in the cell medium as fully processed heterotetramer. Fc fragments released from HIRs-Fc by papain digestion and analyzed by nonreducing SDS-polyacrylamide gel electrophoresis were dimeric. Furthermore, dissociation constants for both chimeras were similar to those for the full-length holoreceptor (wild-type receptor, Kd1 = 200 pM and Kd2 = 2 nM; HIRs-Fc, Kd1 = 200 pM and Kd2 = 40 nM; and HIRs-lambda, Kd1 = 200 pM and Kd2 = 5 nM). These results extend previous observations that dimerization of the membrane-proximal ectodomain is necessary to maintain an intact high-affinity insulin-binding site.

Exon 11 enhances insulin binding affinity and tyrosine kinase activity of the human insulin proreceptor.

Data presented here show that there are significant differences in the insulin binding affinity and the tyrosine kinase activity of the insulin proreceptor isoforms which contain or lack exon 11. The exon 11(+) proreceptor does not show significant variations from the mature processed insulin receptor and has only a 3- to 4-fold reduced affinity for insulin. In contrast, the exon 11(-) proreceptor showed a markedly reduced insulin binding (25- to 50-fold less) when assayed on intact cells. Upon solubilization of the cells, exon 11(-) proreceptor bound insulin with somewhat higher affinity. Mild trypsin treatment of the cells expressing either isoform of the insulin proreceptor restored insulin binding to near-normal levels. Analysis of tyrosine kinase activity revealed that the exon 11(+) proreceptor required somewhat higher concentrations of insulin than the mature processed receptor to achieve maximal autophosphorylation. The exon 11(-) proreceptor failed to fully phosphorylate even at 10(-6) M insulin. Thus, the presence or absence of this short sequence of 12 amino acids affects the folding and/or conformation of the proreceptor so as to confer altered binding of insulin. We suggest that in the absence of exon 11 the proreceptor assumes a strained conformation that disrupts the insulin binding site. Cleavage of the proreceptor at the alpha-beta-subunit junction then allows the alpha-subunit to achieve its normal binding conformation.

Human hypoxanthine-guanine phosphoribosyltransferase deficiency. The molecular defect in a patient with gout (HPRTAshville).

The genetic basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency has been identified by nucleotide sequence analysis of HPRT cDNAs cloned from a patient with gout. A single nucleotide change was identified in two independent clones: an A to G transition at nucleotide 602. Confirmation of a mutation at this site was provided by RNase mapping analysis. The predicted consequence of this transition is an aspartic acid to glycine substitution at amino acid 201. We have designated this variant HPRTAshville. Prior to this report, enzyme activity in HPRTAshville had not been detected by routine assay. Using more sensitive techniques, including an in situ gel assay for HPRT activity, we were able to demonstrate electrophoretic, kinetic, and structural differences between HPRTAshville and normal HPRT. Electrophoretic migration of HPRTAshville has elevated Michaelis constants for 5-phosphoribosyl-1-pyrophosphate and hypoxanthine. Predicted secondary structural alterations may result from the aspartic acid to glycine substitution.

Cross-linking of a B25 azidophenylalanine insulin derivative to the carboxyl-terminal region of the alpha-subunit of the insulin receptor. Identification of a new insulin-binding domain in the insulin receptor.

To identify a site within the insulin receptor ectodomain which forms a binding pocket for B25 Phe and is responsible for initiating conformational changes required for high affinity binding of insulin we have used a novel photoreactive insulin, despentapeptide-(B26-B30) [B25 p-azidophenylalanine-alpha-carboxamide] insulin (APC insulin). This derivative has a highly photoreactive azido group incorporated into the aromatic ring of the B25 phenylalanine amide. APC insulin bound to human insulin receptors overexpressed on a transfected Chinese hamster ovary cell line (P3-A) with an apparent potency of 9-fold relative to that of native insulin and stimulated lipogenesis in rat adipocytes with an average potency equal to porcine insulin. Addition of biotin to the B1 Phe amino group to form despentapeptide-(B26-B30) [B1 (6-biotinylamidocaproyl)phenylalanine B25 p-azidophenylalanine-alpha-carboxamide] insulin derivative (Bio-APC insulin) did not adversely affect receptor-binding affinity and provided a convenient ligand for purification of cross-linked complexes. The efficiency of receptor cross-linking with these reagents was high (70%). To identify the site(s) of cross-linking, the insulin receptor in P3-A cells was first metabolically labeled with various individual 3H-labeled amino acids and then photoaffinity labeled with 125I-Bio-APC insulin, isolated, and digested with Lys-C endoproteinase. The resulting cross-linked peptide fragments were separated by streptavidin-affinity chromatography and sequenced. The smallest identified fragment comprised residues 704-718 of the COOH terminus of the alpha-subunit of the insulin receptor. This B25 Phe cross-linked region of the alpha-subunit lies just upstream of the Exon 11-encoded 12-amino acid COOH-terminal region. Aromatic residues in this predicted alpha-helical region may form a binding pocket for B25 Phe to initiate conformational changes required for stabilizing the high affinity binding state.