Clinical trial: a phase II, randomized study evaluating the safety, pharmacokinetics and anti-viral activity of clevudine for 12 weeks in patients with chronic hepatitis B.

BACKGROUND: Clevudine is a polymerase inhibitor that has the unusual feature of delayed viral rebound after therapy in some patients which may be related to its pharmacokinetics. AIM: To characterize pharmacokinetic and pharmacodynamic profile of clevudine, a potent hepatitis B polymerase inhibitor. METHODS: A multicenter, randomized study comparing 10, 30 and 50 mg clevudine once daily for 12 weeks with 24 weeks off-treatment follow-up. Patients had chronic HBV infection, were nucleoside-naïve without co-infection. HBV viral load (VL) was assayed using Digene Hybrid Capture II with a lower limit of detection of 4700 copies/mL (940 IU/mL). Clevudine levels were measured using a liquid chromatography/mass spectrometery method. RESULTS: A total of 31 patients were enrolled into the 10 mg (n = 10), 30 mg (n = 11) and 50 mg (n = 10) groups, respectively. At week 12, the median VL change was -3.2, -3.7 and -4.2 log(10) copies/mL (-0.64, -0.74 and -0.84 log(10) IU/mL) in the 10, 30 and 50 mg groups, respectively (P = 0.012). At week 12, one of 10, five of 11 and two of 10 patients had VL below the assay lower limit of detection. Clevudine was well tolerated with no severe/serious adverse events. The mean plasma half-life of clevudine was 70 h and consequently is not the cause of the delayed viral rebound seen in some patients. Through modelling, 97% of the maximal treatment effect was reached with a 30 mg daily dose. Six patients had genomic changes without viral rebound. CONCLUSION: Clevudine appears to be a potent and tolerable (over 12 weeks) anti-viral and the optimal dosage appears to be 30 mg once daily.

Overview of the effectiveness of triple combination therapy in antiretroviral-naive HIV-1 infected adults.

AIM: To estimate the effectiveness of triple combination therapy in antiretroviral-naive adults. METHODS: A systematic overview of results from clinical trials involving triple combination therapy with dual nucleoside reverse transcriptase inhibitors (NRTI) and: a protease inhibitor (PI triple); a non-nucleoside reverse transcriptase inhibitor (NNRTI triple); or a third NRTI (triple NUC). Data from 23 clinical trials involving 31 independent treatment groups, 19 unique antiretroviral regimens, and 3257 enrolled patients were included in this study. RESULTS: Median log(10) baseline plasma HIV RNA and CD4 cell count over all trials averaged 4.69 (49,329 copies/ml) and 375 x 10(6) cells/l, respectively. The overall estimated percentage of patients with plasma HIV RNA < or = 400 copies/ml at 24 weeks was 64% [95% confidence interval (CI), 60 to 67%]. The percentages of patients with plasma HIV RNA < or = 50 copies/ml at 48 weeks by drug class were: PI triple, 46% (95% CI, 41 to 52%); NNRTI triple, 51% (95% CI, 43 to 59%); triple NUC, 45% (95% CI, 36 to 54%). The CD4 cell count increase over all trials at 24 and 48 weeks averaged +123 x 10(6) cells/l (95% CI, 111 x 10(6) to 135 x 10(6) cells/l) and +160 x 10(6) cells/l (95% CI, 146 x 10(6) to 175 x 10(6) cells/l), respectively and did not differ between drug classes. In multivariable regression analysis, neither baseline plasma HIV RNA level and CD4 cell count nor treatment regimen predicted plasma HIV RNA < or = 50 copies/ml at week 48. However, pill count was significantly negatively associated with plasma HIV RNA < or = 50 copies/ml at week 48 (P = 0.0085). CONCLUSIONS: The results suggest that three drug regimens containing two NRTI with a PI, a NNRTI, or a third NRTI may provide comparable activity, and practical issues such as daily pill burden should be considered when choosing a treatment regimen.

Site-specific conjugation on serine right-arrow cysteine variant monoclonal antibodies.

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (C(H)3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

Safety assessment, in vitro and in vivo, and pharmacokinetics of emivirine, a potent and selective nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1.

Emivirine (EMV), formerly known as MKC-442, is 6-benzyl-1-(ethoxymethyl)-5-isopropyl-uracil, a novel nonnucleoside reverse transcriptase inhibitor that displays potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity in vivo. EMV showed little or no toxicity towards human mitochondria or human bone marrow progenitor cells. Pharmacokinetics were linear for both rats and monkeys, and oral absorption was 68% in rats. Whole-body autoradiography showed widespread distribution in tissue 30 min after rats were given an oral dose of [(14)C]EMV at 10 mg/kg of body weight. In rats given an oral dose of 250 mg/kg, there were equal levels of EMV in the plasma and the brain. In vitro experiments using liver microsomes demonstrated that the metabolism of EMV by human microsomes is approximately a third of that encountered with rat and monkey microsomes. In 1-month, 3-month, and chronic toxicology experiments (6 months with rats and 1 year with cynomolgus monkeys), toxicity was limited to readily reversible effects on the kidney consisting of vacuolation of kidney tubular epithelial cells and mild increases in blood urea nitrogen. Liver weights increased at the higher doses in rats and monkeys and were attributed to the induction of drug-metabolizing enzymes. EMV tested negative for genotoxic activity, and except for decreased feed consumption at the high dose (160 mg/kg/day), with resultant decreases in maternal and fetal body weights, EMV produced no adverse effects in a complete range of reproductive toxicology experiments performed on rats and rabbits. These results support the clinical development of EMV as a treatment for HIV-1 infection in adult and pediatric patient populations.

Novel benzofuran and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties.

Insulin resistance in the liver and peripheral tissues, together with a pancreatic cell defect, are the common causes of Type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibition of PTPases may be an effective method in the treatment of Type 2 diabetes. We have identified two novel series of benzofuran/benzothiophene biphenyl oxo-acetic acids and sulfonyl-salicylic acids as potent inhibitors of PTP1B with good oral antihyperglycemic activity. To assist in the design of these inhibitors, crystallographic studies have attempted to identify enzyme inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. In the oxo-acetic acid series, hydrophobic substitutents at position-2 of the benzofuran/benzothiophene biphenyl framework interacted with Phe182 of the catalytic site and were very critical to the intrinsic activity of the molecule. The hydrophobic region of the catalytic-site pocket was exploited and taken advantage by hydrophobic substituents at either the alpha-carbon or the ortho aromatic positions of the oxo-acetic acid moiety. Similar ortho aromatic substitutions on the salicylic acid-type inhibitors had no effect, primarily due to the different orientation of these inhibitors in the catalytic site. The most active inhibitors of both series inhibited recombinant human PTP1B with phosphotyrosyl dodecapeptide TRDI(P)YETD(P)Y(P)YRK as the source of the substrate with IC(50) values in the range of 20-50 nM. Compound 68 was one of the most active compounds in vivo, normalizing plasma glucose levels at the 25 mg/kg dose (po) and the 1 mg/kg dose (ip). Compound 68 was also selective against several other PTPases.

Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate.

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.

Conditional, tissue-specific expression of Q205L G alpha i2 in vivo mimics insulin action.

Deficiency of the G protein subunit G alpha i2 that is known to mediate the inhibitory control of adenylylcyclase impairs insulin action [11]. Using the promoter for the phosphoenolpyruvate carboxykinase gene, conditional tissue-specific expression of the constitutively active mutant form (Q205L) of G alpha i2 was achieved in mice harboring the transgene. Expression of Q205L G alpha i2 was detected in liver and adipose tissue of transgenic mice. Whereas the G alpha i2 deficient mice displayed blunted glucose tolerance, the Q205L G alpha i2 expressing mice displayed enhanced glucose tolerance. Hexose transport and the recruitment of GLUT4, but not GLUT1, transporters to the membrane were elevated in adipocytes from Q205L G alpha i2 expressing mice in the absence of insulin. Additionally, hepatic glycogen synthase was found to be activated in Q205L G alpha i2 expressing mice, in the absence of the administration of insulin. Serum insulin levels in transgenic mice fasted overnight were equivalent to those of their control littermates. These data demonstrate that much as G alpha i2 deficiency leads to insulin resistance, expression of Q205L constitutively active G alpha i2 mimics insulin action in vivo, reflecting a permissive role of G alpha i2 in signaling via this growth factor receptor tyrosine kinase linked pathway.

Induction of Galphaq-specific antisense RNA in vivo causes increased body mass and hyperadiposity.

Transgenic BDF-1 mice harboring an inducible, tissue-specific transgene for RNA antisense to Galphaq provide a model in which to study a loss-of-function mutant of Galphaq in vivo. Galphaq deficiency induced in liver and white adipose tissue at birth produced increased body mass and hyperadiposity within 5 weeks of birth that persisted throughout adult life. Galphaq-deficient adipocytes display reduced lipolytic responses, shown to reflect a newly discovered, alpha1-adrenergic regulation of lipolysis. This alpha1-adrenergic response via phosphoinositide hydrolysis and activation of protein kinase C is lacking in the Galphaq loss-of-function mutants in vivo and provides a basis for the increased fat accumulation.

Jun N-terminal kinase mediates activation of skeletal muscle glycogen synthase by insulin in vivo.

Mitogen-activated protein kinases (MAPKs) represent a conserved family of Ser/Thr protein kinases with central roles in intracellular signaling. Activation of three prominent members of the MAPK family, i.e. extracellular response kinases (ERK), jun N-terminal kinase (JNK), and p38, was defined in vivo in order to establish their role, if any, in the cardinal response of skeletal muscle to insulin, the activation of glycogen synthesis. Insulin was found to activate ERK, JNK, and p38 in skeletal muscle. The time courses for activation of the three MAPKs by insulin, however, are distinctly different. Activation of JNK occurs most rapidly, within seconds. Activation of p38 by insulin follows that of JNK, within minutes. Activation of ERK occurs last, 4 min after administration of insulin. The temporal relationship between the activation of ERK, JNK, p38 and the downstream elements p90(rsk) and PP-1 in vivo suggest that JNK, but neither ERK nor p38 MAPKs, mediates insulin activation of glycogen synthase in vivo. Activation of JNK by anisomycin in vivo mimics activation of glycogen synthase by insulin. Challenge by anisomycin and insulin, in combination, are not additive, suggesting a common mode of glycogen synthase activation. The p90(rsk) isoform rapidly activated by insulin is identified as RSK3. In addition, RSK3 can be activated by JNK in vitro. Based upon these data a signal linkage map for activation of glycogen synthase in vivo in skeletal muscle can be constructed in which JNK mediates activation of glycogen synthase via RSK3.

Expression and characterization of human pancreatic preprocarboxypeptidase A1 and preprocarboxypeptidase A2.

We are investigating the potential utility of human carboxypeptidases A in antibody-directed enzyme prodrug therapy (ADEPT). Hybridization screening of a human pancreatic cDNA library with cDNA probes that encoded either rat carboxypeptidase A1 (rCPA1) or carboxypeptidase A2 (rCPA2) was used to clone the human prepro-CPA homologs. After expression of the respective pro-hCPA cDNA in Saccharomyces cerevisiae, the enzymes were purified to homogeneity by a combination of hydrophobic and ion-exchange chromatography. Purified hCPA1 and hCPA2 migrate as a single protein band with M(r) 34,000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Kinetic studies of the purified enzymes with hippuryl-L-phenylalanine resulted in kcat/Km values of 57,000 and 19,000 M-1 s-1 for hCPA1 and hCPA2, respectively. Using the ester substrate, hippuryl-D, L-phenyllactate, we found unique esterase/ peptidase specific activity ratios among hCPA1, hCPA2, rCPA1, and bovine CPA (bCPA) ranging from 13 to 325. Two potential ADEPT substrates, methotrexate-alpha-phenylalanine (MTX-Phe) and methotrexate-alpha-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. The kcat/Km values for MTX-Phe were 440,000 and 90,000 M-1 s-1 for hCPA1 and hCPA2, respectively, and for MTX-naphthylAla these values were 1400 and 1,400,000 M-1 s-1 for hCPA1 and hCPA2, respectively. The kinetic data show that hCPA2 has a larger substrate binding site than the hCPA1 enzyme. Differences between hCPA1 and hCPA2 were also observed in thermal stability experiments at 60 degrees C where the half-life for thermal denaturation of hCPA2 is eightfold longer than that for hCPA1. These experiments indicate that hCPA1 and hCPA2 are potential candidates for use in a human-based ADEPT approach.

Insulin action impaired by deficiency of the G-protein subunit G ialpha2.

Integration of information between tyrosine kinase and G-protein-mediated pathways is necessary, but remains poorly understood. Here we use cells from transgenic mice harbouring inducible expression of RNA antisense to the gene encoding G ialpha2 to show that G ialpha2 is critical for insulin action. G ialpha2 deficiency in adipose tissue and liver produces hyperinsulinaemia, impaired glucose tolerance and resistance to insulin in vivo. Insulin resistance affects glucose-transporter activity and recruitment, counterregulation of lipolysis, and activation of glycogen synthase, all of which are cardinal responses to insulin. G ialpha2 deficiency increases protein-tyrosine phosphatase activity and attenuates insulin-stimulated tyrosine phosphorylation of IRS (insulin-receptor substrate 1) in vivo. G ialpha2 deficiency creates a model for insulin resistance characteristic of noninsulin-dependent diabetes mellitus (NIDDM), implicating G ialpha2 as a positive regulator of insulin action.

Suppression of Gi alpha 2 enhances phospholipase C signalling.

G-proteins mediate transmembrane signalling from a populous group of cell-surface receptors to a smaller group of effectors that includes adenylate cyclase, various ion channels and phospholipase C. Stem cells (F9 teratocarcinoma) or rat osteosarcoma 17/2.8 cells in which Gi alpha 2 expression is abolished by antisense RNA display markedly elevated basal inositol 1,4,5-trisphosphate accumulation and a potentiated phospholipase C response to stimulatory hormones. Expression of the Q205L mutant of Gi alpha 2, which is constitutively active, was found to block persistently hormonally stimulated phospholipase C activity, implicating Gi alpha 2 as an inhibitory regulator of phospholipase C signalling. Analysis using Gi alpha 2-deficient adipocytes of transgenic mice provided further evidence for a role for Gi alpha 2 in phospholipase C regulation, demonstrating in vivo that loss of Gi alpha 2 elevates basal, and markedly potentiates hormonally stimulated, phospholipase C activity. This report demonstrates for the first time that a single G-protein, G12, can regulate two distinct signalling pathways, i.e. adenylate cyclase and phospholipase C.

Induction of G alpha i2-specific antisense RNA in vivo inhibits neonatal growth.

Guanosine triphosphate-binding regulatory proteins (G proteins) are key elements in transmembrane signaling and have been implicated as regulators of more complex biological processes such as differentiation and development. The G protein G alpha i2 is capable of mediating the inhibitory control of adenylylcyclase and regulates stem cell differentiation to primitive endoderm. Here an antisense RNA to G alpha i2 was expressed in a hybrid RNA construct whose expression was both tissue-specific and induced at birth. Transgenic mice in which the antisense construct was expressed displayed a lack of normal development in targeted organs that correlated with the absence of G alpha i2. The loss of G alpha i2 expression in adipose tissue of the transgenic mice was correlated with a rise in basal levels of adenosine 3',5'-monophosphate (cAMP) and the loss of receptor-mediated inhibition of adenylylcyclase. These data expand our understanding of G protein function in vivo and demonstrate the necessity for G alpha i2 in the development of liver and fat.

Gi alpha 2 mediates the inhibitory regulation of adenylylcyclase in vivo: analysis in transgenic mice with Gi alpha 2 suppressed by inducible antisense RNA.

The role of the GTP-binding regulatory protein (G-protein) Gi alpha 2 in vivo was explored using transgenic mice in which the alpha-subunit of Gi alpha 2 was suppressed by antisense RNA. Rat hepatoma FTO-2B cells provide an ideal test system for constructs employing the expression vector pPCK-AS, designed to express antisense RNA at birth under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Cells transfected with the expression vector containing a sequence antisense to Gi alpha 2 (pPCK-ASGi alpha 2) displayed expression of RNA antisense to Gi alpha 2 that, like transcription of the PEPCK gene, was inducible by cyclic AMP. Expression of RNA antisense to Gi alpha 2 and suppression of the expression of Gi alpha 2, but not Gsa and Gi alpha 3, was observed in the transfected FTO-2B cells. BDF1 mice carrying the transgene displayed suppression of Gi alpha 2 in liver and fat, two targets for tissue-specific expression of the PEPCK gene. The loss of Gi alpha 2 in white adipocytes of transgenic mice resulted in 3.1-fold elevation of basal cyclic AMP accumulation. Cyclic AMP accumulation in response to stimulation by epinephrine (10 microM) was normal in adipocytes of transgenic mice, demonstrating no alteration in the stimulatory adenylylcyclase capacity in the Gi alpha 2-deficient cells. The inhibitory adenylylcyclase pathway, in sharp contrast, was severely blunted in response to challenge by the inhibitory A1-purinergic agonist, (-)R-N6-phenylisopropyladenosine. These studies illuminate a critical role of Gi alpha 2 in the inhibitory adenylylcyclase signaling pathway in vivo.

Insulin like growth factor-I induces limited association of phosphatidylinositol 3-kinase to its receptor.

Stimulation by insulin-like growth factor-I (IGF-I) of LISN C4 cells, a mouse fibroblast cell line that overexpresses human IGF-I receptors, led to an increase in the amount of a phosphatidylinositol kinase that could be immunoprecipitated by anti-IGF-I receptor or anti-phosphotyrosine antibodies. The identity of the lipid produced in phosphatidylinositol kinase assays of anti-IGF-I receptor or anti-phosphotyrosine immunoprecipitates indicated that IGF-I selectively increased the amount of immunoprecipitated phosphatidylinositol 3-kinase activity. The amount of immunoprecipitated phosphatidylinositol 3-kinase activity that was increased by IGF-I followed a time course that paralleled the stimulation of IGF-I receptor beta-subunit autophosphorylation. The amount of phosphatidylinositol 3-kinase activity detected in anti-IGF-I receptor immunoprecipitates represented only 2% of that which was immunoprecipitated by anti-phosphotyrosine antibody. Furthermore, phosphatidylinositol 3-kinase activity which was recovered with anti-phosphotyrosine antibody was present in both cytosol and particulate cell fractions at approximately similar levels. Taken together, these results suggest that the stimulation of the IGF-I receptor tyrosine kinase leads to an increase in the amount of phosphatidyl inositol 3-kinase activity immunoprecipitated by antiphosphotyrosine and anti-IGF-I receptor antibodies and to a limited association with the IGF-I receptor itself, even though these cells express very high levels of IGF-I receptors. That the majority of phosphatidylinositol 3-kinase activity does not tightly associate with the IGF-I receptor after IGF-I stimulation suggests that it may be associated with other tyrosine phosphorylated proteins. Alternatively, the kinase itself may become phosphorylated on tyrosine and dissociate from the IGF-I receptor. In this manner, an increase of phosphatidylinositol 3-kinase activity by IGF-I deviates from the activation of phosphatidylinositol 3-kinase by platelet-derived growth factor receptor in that a tight association with the receptor is not produced after stimulation.

Insulin/IGF-I receptor hybrids: a mechanism for increasing receptor diversity.

Insulin and IGF-I receptors are homologous disulfide linked alpha 2 beta 2 tetramers. These tetramers are formed biosynthetically when proreceptors containing alpha and beta subunits in a single uninterrupted linear peptide form disulfide linked homodimers and are subsequently proteolytically cleaved at the alpha-beta junctions. Cells expressing both receptors also express hybrid receptors that contain one insulin receptor alpha and beta subunit, and one IGF-I receptor alpha and beta subunit. These presumably form by the association of mixed proreceptors. Hybrid receptors greatly expand the possible repertoire of cellular responses to hormonal stimulation. Although not yet examined in detail, both the hormone binding and the signaling properties of the hybrid receptor appear to be different from that of either insulin or IGF-I receptor. Regulatory mechanisms that involve either insulin or IGF-I receptor, at the level of expression or subsequently, could alter the expression or function of the hybrid receptor or the other receptor. Similarly, pathology in one receptor could affect both the hybrid and other receptor, or perhaps be partially compensated for by a hybrid receptor. The magnitude of these effects could vary greatly in different tissues depending upon the relative level of expression of the different receptor forms. These postulated responses might explain some of the complex heterogeneity and linkage of these receptors that have been observed previously.

Hybrid receptors.

Many hormone and growth factor receptors form homomeric oligomers. Subunits of related receptors can be mixed to form hybrid receptors. These can have novel ligand-binding and signaling properties. The additional flexibility that results from the ability to independently regulate the expression and function of different component subunits greatly increases the spectrum of cellular responses to extracellular signals.

Insulin-like growth factor I receptor beta-subunit heterogeneity. Evidence for hybrid tetramers composed of insulin-like growth factor I and insulin receptor heterodimers.

In both NIH3T3 cells and HepG2 cells, insulin-like growth factor I (IGF-I) receptors possess two beta-subunits that display different electrophoretic mobilities. Increasing concentrations of IGF-I stimulated the phosphorylation of both beta-subunits to a similar extent, whereas insulin stimulated the phosphorylation of both subunits only at elevated concentrations. Both beta-subunits were immunoprecipitated with p5, an insulin receptor-specific anti-peptide antibody, or with A410, a polyclonal anti-insulin receptor antisera. However, if the tetrameric IGF-I receptor was first dissociated into alpha-beta heterodimers with 1 mM dithiothreitol, only the lower molecular weight beta-subunit was immunoprecipitated. These results suggested that p5 and A410 specifically recognized the lower molecular weight beta-subunit but immunoprecipitated the higher molecular weight beta-subunit because it was present in the same disulfide linked tetramer. Similarly, alpha-IR-3, an antibody specific for the alpha-subunit of the IGF-I receptor, immunoprecipitated both types of beta-subunit from the intact tetramer but only the higher molecular weight beta-subunit from the dissociated heterodimers, suggesting that there are two types of alpha-subunits in the same tetramer and that the alpha-subunit recognized by alpha-IR-3 is only associated with the higher molecular weight beta-subunit. Tryptic phosphopeptide maps of the lower molecular weight beta-subunit of IGF-I receptor were different from the higher molecular weight beta-subunit, but were similar to those of the insulin receptor beta-subunit. Thus, by immunochemical cross-reactivity and structural criteria, the lower molecular weight beta-subunit of the IGF-I receptor was similar to the beta-subunit of insulin receptor. These data suggest that there exists a species of IGF-I receptor that is a hybrid composed of an insulin receptor alpha-beta heterodimer and an IGF-I receptor alpha-beta heterodimer. The existence of such a hybrid receptor could have important functional consequences.

Beta-adrenergic receptors display intramolecular disulfide bridges in situ: analysis by immunoblotting and functional reconstitution.

The molecular nature of mammalian beta-adrenergic receptors in situ was probed using immunoblotting and functional reconstitution techniques. Membrane proteins of cells replete with beta-adrenergic receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose and then probed with anti-receptor antibodies. When cell membranes were first treated with agents that cleave disulfides of proteins, immunoblots of these membranes revealed intense immunoreactive bands with electrophoretic mobility similar to that of protein standards of Mr 65,000-67,000, comigrating with purified, reduced, and alkylated beta-adrenergic receptors. However, when cell membranes were prepared under anaerobic conditions, solubilized in the presence of agents that alkylate thiols, and denatured in the absence of added thiols, immunoblotting revealed receptor with Mr 55,000, rather than 65,000. This faster electrophoretic mobility is associated with the presence of intramolecular disulfides in the purified receptor and demonstrates that beta-adrenergic receptors possess intramolecular disulfide bridges in situ. Purified receptors that demonstrate this faster mobility (Mr 55,000 under nonreducing conditions) were co-reconstituted into phospholipid vesicles with the stimulatory GTP-binding protein GS and their ability to catalyze the binding of [35S]guanosine-5'-O-(3-thio)triphosphate to GS was measured. Agonist (isoproterenol) as well as thiol increased the receptor-promoted activation of GS. Taken together, these data demonstrate that native beta-adrenergic receptors possess one or more intramolecular disulfide bridges in situ, reduction of which causes functional activation of the receptor.