Regulation of the insulin receptor by a monoclonal anti-receptor antibody. Evidence that receptor down regulation can be independent of insulin action

In the present study, we investigated the ability of a monoclonal antibody to the insulin receptor to regulate the expression of the insulin receptor of IM-9 lymphocytes. Previously, this antibody was shown to be a competitive antagonist of insulin action on severe metabolic functions. In the present study, we report that preincubation of IM-9 cells with the monoclonal antibody caused a dose- and time-dependent decrease in the subsequent ability of these cells to bind 125I-insulin, a phenomenon termed down regulation. The antibody was approximately 100 times more potent than insulin at down regulating the receptor. In contrast, the antibody was 5 times less potent than insulin in competing for binding to insulin receptors and dissociated 4 times more rapidly than insulin from IM-9 cells. Three lines of evidence suggested that the mechanism of down regulation by the antibody was the same as the one used by insulin. First, both agents caused a rapid initial decrease in insulin binding to cells followed by a slower, gradual decrease in binding. Second, the down regulation caused by both was reversible, and this reversibility required new protein synthesis. Third, the antibody, like insulin, accelerated receptor degradation. Since the antibody does not mimic the other effects of insulin on metabolic processes, these results suggest that the mechanism of insulin receptor down regulation is different from the mechanism of insulin action on other cellular functions.     

Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations

The melanocortin-3 receptor (MC3R) is a member of family A rhodopsin-like G protein-coupled receptors. Mouse genetic studies suggested that MC3R and the related MC4R are non-redundant regulators of energy homeostasis. Lack of Mc3r leads to higher feed efficiency and fat mass. However, until now only a few MC3R mutations have been identified in humans and the role of MC3R in the pathogenesis of obesity was unclear. In the present study, we performed detailed functional studies on nine naturally occurring MC3R mutations recently reported. We found that all nine mutants had decreased cell surface expression. A260V, M275T, and L297V had decreased total expression whereas the other six mutants had normal total expression. Mutants S69C and T280S exhibited significant defects in ligand binding and signaling. The dramatic defects of T280S might be partially caused by decreased cell surface expression. In addition, we found mutants M134I and M275T had decreased maximal binding but displayed similar signaling properties as wild-type MC3R. All the other mutants had normal binding and signaling activities. Co-expression studies showed that all mutants except L297V did not affect wild-type MC3R signaling. Multiple mutations at T280 demonstrated the necessity of Thr for cell surface expression, ligand binding, and signaling. In summary, we provided detailed data of these novel human MC3R mutations leading to a better understanding of structure-function relationship of MC3R and the role of MC3R mutation in obesity.     

Insulin action on adipocytes. Evidence that the anti-lipolytic and lipogenic effects of insulin are mediated by the same receptor.

1. The dose-response relationships of insulin stimulation of lipogenesis and inhibition of lipolysis were studied simultaneously by using rat adipocytes to determine whether these different effects of insulin are mediated through the same or different sets of receptors. 2. The sensitivity (defined as the concentration of insulin required to produce a half-maximal effect) of the stimulated lipogenic response to insulin was not significantly different from the sensitivity of the anti-lipolytic response to insulin. The addition of different adrenaline and glucose concentrations did not alter the half-maximal concentration of insulin required to inhibit lipolysis. 3. The specificities of the lipogenic and antilipolytic responses were studied by using insulin analogues. The sensitivities of the lipogenic and anti-lipolytic responses were the same for five chemically modified insulins and hagfish insulin, which have potencies compared with bovine insulin of between 3 and 90%. 4. Starving rats for 48h significantly increased the sensitivities of both the antilipolytic and lipogenic responses to insulin, but the changes in the sensitivities of both lipogenesis and anti-lipolysis returned to that of fed rats. 5. We conclude that insulin stimulates lipogenesis and inhibits lipolysis over the same concentration range. These observations provide powerful evidence that the different effects of insulin are mediated through the same set of receptors.     

Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation.

Some patients with extreme insulin resistance have mutations in their insulin receptor gene. We previously identified five such mutations located in the extracellular domain of the insulin receptor (Asn-->Lys15, His-->Arg209, Phe-->Val382, Lys-->Glu460, and Asn-->Ser462) and studied the effects of these mutations upon posttranslational processing, insulin binding, and tyrosine autophosphorylation. We now characterize the ability of these mutant receptors to mediate biological actions of insulin in transfected NIH-3T3 fibroblasts. All cell lines expressing mutant receptors showed marked impairment in insulin-stimulated c-jun expression and thymidine incorporation when compared with cells expressing wild-type human insulin receptors. The most severe impairment was seen in cells expressing the Val382 mutant (a mutation which causes an intrinsic defect in receptor autophosphorylation). These cells had insulin responses similar to the untransfected cells (used as a negative control). In contrast, cells expressing the Lys15 mutant have the ability to achieve a normal level of maximal autophosphorylation but require an abnormally high concentration of insulin to do so (as the result of decreased insulin binding affinity). These cells show a higher basal rate and much lower insulin stimulation of both c-jun expression and thymidine incorporation when compared with the cells expressing the wild-type human insulin receptors. This pattern is also seen in the cells expressing the other mutants with normal autophosphorylation (Arg209, Glu460, and Ser462). Although the most severe defects in insulin action are seen with the mutation which has an intrinsic defect in receptor autophosphorylation, the ability to undergo normal autophosphorylation does not seem to preclude mutations from impairing the ability of receptors to mediate some of the actions of insulin.     

Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor

Recently, we have described a COOH-terminal deletion mutation of the human insulin receptor (HIR delta CT) that exhibits normal insulin-mediated kinase activity and endocytosis, but is inefficient in stimulating glucose transport and glycogen synthase (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J.M. (1988) J. Biol. Chem. 263, 8904-8911; Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this paper, we report that despite this defect in metabolic signaling, the truncated receptor exhibits augmented mitogenic activity compared to normal receptors. These results were verified in three independently isolated clones of Rat 1 fibroblasts transfected with the HIR delta CT cDNA. The increase in insulin sensitivity of mitogenic stimulation was proportional to the number of HIR delta CT receptors expressed on the cells. By contrast, only the cells with normal receptors and none of the HIR delta CT clones exhibit increased sensitivity for a metabolic action of insulin, the stimulation of glucose uptake. Stimulation of cells by other mitogens and autoradiographic analysis confirm that the enhanced mitogenic effects seen in HIR delta CT cells are attributable only to the presence of the truncated insulin receptors. These receptors mediate the tyrosine phosphorylation of a number of cellular proteins, and the pattern of these phosphorylations differs quantitatively from that seen in cells with normal receptors. We conclude: 1) The COOH terminus plays a role in signaling metabolic actions of insulin, perhaps through its recognition of substrates for the receptor kinase. 2) By contrast, the COOH terminus is an inhibitory regulator of mitogenesis, and removal of the terminal 43 amino acids converts the receptor from a moderately active growth signaler to a very active one. 3) The changes seen in biologic activities of the HIR delta CT receptor are associated with quantitative changes in substrate phosphorylation by the receptor kinase.     

Evidence that vascular actions of PHI are mediated by a VIP-preferring receptor

Vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are homologous neuropeptides which share vasodilatory properties. This paper addresses the question of whether PHI exerts its vascular action via a receptor distinct from that for VIP. Radioligand binding experiments were done using [Tyr(125I)10]VIP, [Tyr(125I)22]porcine PHI, [Tyr(125I)10]rat PHI and arterial preparations from rat, bovine and porcine species. The radioiodination of rat PHI by the lactoperoxidase-glucose oxidase method and analysis of the structure of the major radiolabeled derivatives were described. All the receptor binding experiments identified a VIP-preferring receptor irrespective of which radioligand or arterial preparation was utilized. VIP and PHI peptides demonstrated cross-desensitization in studies of relaxation of porcine coronary arterial strips in vitro. The present results favor the conclusion that the vascular actions of the PHI peptides are best explained by binding to a VIP-preferring receptor.     

VH sequence of a human anti-Sm autoantibody. Evidence that autoantibodies can be unmutated copies of germline genes

The utilization of germline genes for the synthesis of autoantibodies has been suspected for many years based on the presence of cross-reactive idiotypes among patients as well as in some healthy first-degree relatives of patients with several autoimmune diseases including SLE. One such system of idiotypes involves anti-Sm antibodies, which are highly specific for SLE. To definitively establish the utilization of germline genes in the Sm system, we produced human-human B cell hybridomas from a patient with SLE who had circulating anti-Sm antibodies. One stable hybridoma designated 4B4 secretes an IgM-kappa mAb that binds Sm and shares idiotypic determinants with other anti-Sm antibodies. A second anti-Sm antibody (3C3), isolated from the same patient was also studied. Oligo(dT) priming was used to produce cDNA corresponding to full length IgM. Sequence analysis revealed that the VH gene segment (1-96) of 4B4 is identical to a VH sequence previously detected in a fetal liver cDNA library by Schroeder and his co-workers as well as a germline VH recently described by Berman and his associates. The identity of a lupus mAb and sequences derived from unrelated individuals provides strong evidence that this autoantibody is a direct copy of a germline gene.     

Structural effects of naturally occurring human blood group B galactosyltransferase mutations adjacent to the DXD motif

Human blood group A and B antigens are produced by two closely related glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) utilizes UDP-GalNAc to extend H antigen acceptors (Fuc alpha(1-2)Gal beta-OR) producing A antigens, whereas a galactosyltransferase (GTB) utilizes UDP-Gal as a donor to extend H structures producing B antigens. GTA and GTB have a characteristic (211)DVD(213) motif that coordinates to a Mn(2+) ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the (211)DVD(213) motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the B(el) variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to A(weak)B phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in k(cat) compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn(2+) was disrupted by Arg-214 causing displacement of the metal by a water molecule. Kinetic characterizations of the M214T and M214V mutants revealed they both had GTA and GTB activity consistent with the serology. The crystal structure of the M214T mutant showed no change in DVD coordination to Mn(2+). Instead a critical residue, Met-266, which is responsible for determining donor specificity, had adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity.     

Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin

Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.     

Functional properties of a naturally occurring Trp1200----Ser1200 mutation of the insulin receptor

Based on the sequence of cDNA encoding the intracellular domain of the insulin receptor beta-subunit, we recently defined a heterozygous point mutation causing a Ser for Trp substitution at position 1200 in the tyrosine kinase domain of a patient (BI-2) with the type A syndrome of insulin resistance. We have now sequenced the remainder of BI-2's insulin receptor cDNA-coding region and find no additional alterations in the encoded proreceptor protein. The nucleotide sequence of cDNA encoding the portion of the beta-subunit which includes Trp1200 was normal in BI-2's unaffected mother. Hybridization of a mutant allele-specific oligonucleotide to polymerase chain reaction-amplified cDNA confirmed the presence of the mutant allele in the proband and excluded it in her unaffected sister and mother, 18 normal control subjects, and six other subjects with insulin resistance. To determine whether this mutation had functional consequences for receptor signalling, we reconstructed it into a full-length insulin receptor cDNA expression vector. Chinese hamster ovary cells were transfected with mutant cDNA, and the expressed insulin receptors were compared to receptors expressed by cells transfected with wild-type receptor cDNA. Both mutant and wild-type receptors were properly processed into receptor alpha- and beta-subunits, were expressed on the cell surface, and displayed similar insulin-binding affinity. In contrast, insulin-stimulated autophosphorylation of the mutant receptors was severely impaired, whether assessed in intact cells or with a partially purified receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered expression of the two naturally occurring human insulin receptor variants in isolated adipocytes of non-insulin-dependent diabetes mellitus patients.

The human insulin receptor gene is expressed in two variant isoforms which differ by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the extracellular alpha-subunit as a consequence of alternative splicing of exon 11. Expression of the two variant isoforms is regulated in a tissue-specific manner. In this study, we have measured the levels of the two receptor variants in isolated adipocytes from 10 non-insulin-dependent diabetes mellitus (NIDDM) and 11 normal subjects using an immunological assay, based on the ability of a human anti-receptor autoantibody to discriminate between HIR-A and HIR-B. Results indicate that levels of HIR-B variant are increased in NIDDM patients.     

Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites.

We have recently characterized a mutant insulin receptor (Y/F2) in which the two tyrosines in the carboxyl terminus (Tyr1316, Tyr1322) were mutated to phenylalanine. Compared with wild type receptors, the Y/F2 receptor exhibited markedly enhanced sensitivity to insulin-stimulated DNA synthesis with normal insulin-stimulated glucose uptake (Takata, Y., Webster, N. J. G., and Olefsky, J. M. (1991) J. Biol. Chem. 266, 9135-9139). In this paper, we present further evidence for the divergence of the metabolic and mitogenic signaling pathways utilized by the insulin receptor. The mutant receptor showed normal sensitivity and responsiveness for insulin-stimulated glucose incorporation into glycogen. The insulin sensitivity for phosphorylation of two substrates (pp180 and pp220) was the same in both Y/F2 cells and HIRc cells. Phosphotyrosine content, however, was greater in Y/F2 cells than in HIRc cells, especially in the basal state. Insulin stimulated S6 kinase activity 2-6-fold, with an ED50 of -10 nM in Rat 1 cells and 0.5 nM in HIRc cells. The sensitivity to insulin was enhanced in Y/F2 cells with an ED50 of 0.1 nM. These effects were insulin-specific, since insulin-like growth factor (IGF)-I-stimulated mitogenesis was normal. In summary: 1) Y/F2 receptors exhibit normal metabolic and enhanced mitogenic signaling; 2) the enhanced mitogenic signaling is specific for the insulin receptor in the Y/F2 cells, since IGF-I-stimulated mitogenesis is normal; 3) Y/F2 cells display increased endogenous substrate phosphorylation and augmented insulin-stimulated S6 kinase activity placing these responses among insulin's mitogenic effects; and 4) these results are consistent with the concept that the COOH-terminal tyrosine residues of the insulin receptor are normally inhibitory to mitogenic signaling.     

Immunostimulatory potential of silencing RNAs can be mediated by a non-uridine-rich toll-like receptor 7 motif

Microbial infections trigger a multiplicity of responses in the host via innate immune sensors, including the Toll-like receptors (TLRs). TLR7 and TLR8, located in endosomes, detect pathogen-derived RNA, which can be mimicked by synthetic single-stranded oligoribonucleotides (ORNs). Detailed analysis of the immunostimulatory properties of numerous silencing RNAs (siRNAs) revealed that almost all tested siRNAs with a phosphodiester backbone actively stimulated cytokine production in human peripheral blood immune cells, but not all of them did contain previously described guanosine/uridine TLR7 or adenosine/uridine TLR8 motifs. By analysis of sequence variants of these siRNAs (as single- or double-strands), we were able to identify a new immunostimulatory, non-uridine-rich TLR7 motif that is present in many published siRNAs. Interestingly, the activity of this motif is dependent on the backbone chemistry. Phosphorothioate ORNs containing the motif did not stimulate immune activation, whereas phosphodiester ORNs of the same sequence induced a strong TLR7-biased immune response with high amounts of interferon-alpha. Using TLR7- and Myd88-deficient mice, we demonstrated that stimulation by ORNs containing this motif was TLR7 dependent. Our findings are of therapeutic relevance as this motif is present in many siRNA sequences and will to contribute to the immunostimulatory properties of unmodified siRNAs.     

Gin mutants that can be suppressed by a Fis-independent mutation.

The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites. Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence. We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding. Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype. Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis. The corresponding mutation, M to I at position 108 (M108I), is located in a putative alpha-helical structure, which in the homologous gamma delta resolvase has been implicated in dimerization. The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction. The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion. The corresponding mutations are located in different domains of the protein. The properties of these mutants in connection with the role of Fis in inversion will be discussed.     

Stimulation of phospholipase C activity by insulin is mediated by both isotypes of the human insulin receptor.

The human insulin receptor exists in two isoforms, HIR-A and HIR-B. We studied whether both insulin receptor isotypes are able to mediate an insulin signal to phospholipase C. Plasma membranes were prepared from rat-1 fibroblasts transfected either with HIR-A or HIR-B and insulin stimulated PIP-hydrolysis was determined. We found that insulin stimulates PIP-hydrolysis in a similar dose dependent manner and to a similar extent in plasma membranes expressing HIR-A and HIR-B. These data suggest that both receptor isoforms are equally able to activate phospholipase-C.     

Evidence that activation of acetyl-CoA carboxylase by insulin in adipocytes is mediated by a low-Mr effector and not by increased phosphorylation.

The activation of acetyl-CoA carboxylase (measured in a crude supernatant fraction) caused by insulin treatment of adipocytes was completely unaffected by the addition of a large amount of highly purified protein phosphatase to the supernatant fraction. Under the same conditions the inhibition of acetyl-CoA carboxylase by adrenaline was totally reversed. Experiments with 32P-labelled adipocytes showed that insulin increased the total phosphorylation of acetyl-CoA carboxylase from 2.7 to 3.5 molecules of phosphate/240 kDa subunit, and confirmed that this increase was partially accounted for by phosphorylation within a specific peptide (the 'I-site' peptide). Protein phosphatase treatment of the crude supernatant fractions removed over 80% of the 32P radioactivity from the enzyme and removed all detectable radioactivity from the I-site peptide. The effect of insulin on acetyl-CoA carboxylase activity, but not the effect on phosphorylation, was lost on purification of the enzyme on avidin-Sepharose. The effect on enzyme activity was also lost if crude supernatant fractions were subjected to rapid gel filtration after treatment under conditions of high ionic strength, similar to those used in the avidin-Sepharose procedure. These results show that, although insulin does increase the phosphorylation of acetyl-CoA carboxylase at a specific site, this does not cause enzyme activation. They suggest instead that activation of the enzyme by insulin is mediated by a tightly bound low-Mr effector which dissociates from the enzyme at high ionic strength.     

Activation of phosphatidylinositol-3-kinase by insulin is mediated by both A and B human insulin receptor types.

Activation of a phosphatidylinositol-3-kinase (PI-3-kinase) is one of the earliest consequences of insulin binding to the receptor. The human insulin receptor exists in two isoforms which differ in the length of the alpha-subunit (HIR-A = 719 aa, HIR-B = 731 aa). To test whether both isoforms transduce an insulin signal on PI-3-kinase we used rat-1-fibroblasts expressing HIR-A or HIR-B. We found that insulin stimulates 32P incorporation into PIP through both HIR-A and HIR-B to a similar extent (approx. 8-10 fold).     

Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant human melanocortin-4 receptors

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor that plays an essential role in regulating energy homeostasis. Defects in MC4R are the most common monogenic form of obesity, with about 170 distinct mutations identified in human. In addition to the conventional G_s-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2). Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the G_s-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2-fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the G_s-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.