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J M Backer

Publications and source records attributed to J M Backer.

At least 55 records · Page 3Linked to original sources
PubMed1993

Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding.

We have developed a competition binding assay to quantify relative affinities of isolated Src-homology 2 (SH2) domains for phosphopeptide sequences. Eleven synthetic 11-12-amino acid phosphopeptides containing YMXM or YVXM recognition motifs bound to a PI 3-kinase p85 SH2 domain with highest affinities, including sequences surrounding phosphorylated tyrosines of the PDGF, CSF-1/c-Fms, and kit-encoded receptors, IRS-1, and polyoma middle T antigens; matched, unphosphorylated sequences did not bind. A scrambled YMXM phosphopeptide or sequences corresponding to the GAP or PLC-gamma SH2 domain binding motifs of the PDGF, FGF, and EGF receptors bound to the p85 SH2 domain with 30-100-fold reduced affinity, indicating that this affinity range confers specificity. Binding specificity was appropriately reversed with an SH2 domain from PLC-gamma: a phosphopeptide corresponding to the site surrounding PDGF receptor Tyr1021 binds with approximately 40-fold higher affinity than a YMXM-phosphopeptide. We conclude that essential features of specific phosphoprotein/SH2 domain interactions can be reconstituted using truncated versions of both the phosphoprotein (a phosphopeptide) and cognate SH2 domain-containing protein (the SH2 domain). SH2 domain binding specificity results from differences in affinity conferred by the linear sequence surrounding phosphotyrosine.

Amino Acid Sequence↗
PubMed1993

BpaB25 insulins. Photoactivatable analogues that quantitatively cross-link, radiolabel, and activate the insulin receptor.

Benzoylphenylalanine (Bpa), a photoactivatable amino acid was incorporated into the 25 position of the insulin B-chain by a combination of chemical synthesis and enzymatic semisynthesis. BpaB25 insulin binds specifically to the insulin receptor with an affinity approximately 40% that of native insulin. Addition of biotin or epsilon-amino-hexanoic acid-iminobiotin at the epsilon-LysB29 position to form BpaB25,B29 epsilon-biotinyl (BBpa) or BpaB25,B29 epsilon-(Aha-iminobiotin) analogues had little adverse effect on receptor binding affinity and provided a convenient handle for affinity purification, gel shift assays, and blotting of cross-linked complexes with avidin. The analogues were readily 125I-iodinated with the majority of 125I being incorporated at the TyrA14 position; the monoiodo-A14 derivative was easily separated from other forms by high performance liquid chromatography. Photolysis of a complex of the insulin receptor and either BpaB25 insulin or [125I]iodo-TyrA14,BpaB25 insulin yields a covalent insulin-receptor complex. The efficiency of cross-linking with these reagents was unusually high, ranging from 60 to 100%. Furthermore, cross-linking to the insulin receptor results in kinase activation in vitro, and in intact cells insulin receptor phosphorylation and internalization were both activated. Notably, even at saturating concentrations one molecule of BpaB25 insulin covalently cross-linked each alpha 2 beta 2 receptor, demonstrating that holoreceptor activation occurs with one high affinity insulin binding site occupied and, if a second insulin binds with lower affinity it must be in a different orientation. Bpa insulin analogues form a new class of photoaffinity reagents which facilitate studies relating insulin-insulin receptor structure and function.

Affinity Labels↗
PubMed1993

The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling.

GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homologue of the Caenorhabditis elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases. In this report we show that in response to insulin, GRB2 forms a stable complex with two tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc. Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, we have identified a short sequence motif (YV/IN) present in IRS-1, EGFR and Shc, which specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could provide the core for a large signalling complex. We propose a model whereby insulin stimulation leads to formation of multiple protein--protein interactions between GRB2 and the two targets IRS-1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase.

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.

Amino Acid Sequence↗
PubMed1993

Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus.

Insulin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which in turn binds to and activates phosphatidylinositol 3-kinase (PI 3-kinase). In the present study, we have examined these processes in animal models of insulin-resistant and insulin-deficient diabetes mellitus. After in vivo insulin stimulation, there was a 60-80% decrease in IRS-1 phosphorylation in liver and muscle of the ob/ob mouse. There was no insulin stimulation of PI 3-kinase (85 kD subunit) association with IRS-1, and IRS-1-associated PI 3-kinase activity was reduced 90%. Insulin-stimulated total PI 3-kinase activity was also absent in both tissues of the ob/ob mouse. By contrast, in the streptozotocin diabetic rat, IRS-1 phosphorylation increased 50% in muscle, IRS-1-associated PI 3-kinase activity was increased two- to threefold in liver and muscle, and there was a 50% increase in the p85 associated with IRS-1 after insulin stimulation in muscle. In conclusion, (a) IRS-1-associated PI 3-kinase activity is differentially regulated in hyperinsulinemic and hypoinsulinemic diabetic states; (b) PI 3-kinase activation closely correlates with IRS-1 phosphorylation; and (c) reduced PI 3-kinase activity may play a role in the pathophysiology of insulin resistant diabetic states, such as that seen in the ob/ob mouse.

Animals↗
PubMed1993

IRS-1 is a common element in insulin and insulin-like growth factor-I signaling to the phosphatidylinositol 3'-kinase.

IRS-1 is a unique cytosolic protein that becomes tyrosine phosphorylated during insulin stimulation of intact cells and immediately associates with the phosphatidylinositol 3'-kinase (PtdIns 3'-kinase). The insulin-like growth factor-I (IGF-I) receptor also mediated the tyrosine phosphorylation of IRS-1 and increased the amount of PtdIns 3'-kinase activity bound to IRS-1 in Chinese hamster ovary cells. Purified insulin receptor and IGF-I receptor phosphorylated recombinant baculovirus-produced IRS-1 on similar sites in vitro, and phosphorylated baculovirus-produced IRS-1 bound PtdIns 3'-kinase activity from lysates of quiescent cells. Treatment of cells with IGF-I activated the PtdIns 3'-kinase, suggesting that IGF-I activates the PtdIns 3'-kinase through IRS-1 binding to p85 in a manner similar to insulin. Chinese hamster ovary cells overexpressing IRS-1 demonstrated increased tyrosine phosphorylation of IRS-1, and more PtdIns 3'-kinase activity associated with IRS-1 in these cells. These data demonstrate that IRS-1 is a common element for signal transmission by the IGF-I and insulin receptors.

Animals↗
PubMed1993

Chromosomal localization and nucleoside diphosphate kinase activity of human metastasis-suppressor genes NM23-1 and NM23-2.

Human metastasis-suppressor genes nm23-1 (NME1) and nm23-2 (NME2) are implicated in control of the metastatic potential of malignant cells. Using somatic cell hybrid analysis and fluorescence in situ hybridization we co-localized both genes to 17q21.3. The 17q21 region carries the locus responsible for early-onset familial breast-ovarian cancer and several other genes that are involved in tumorigenesis and differentiation and undergo frequent rearrangements during neoplastic development. Thus, our mapping places the NME genes in a region that may be subjected to multiple selection pressures. NME1 and NME2 genes were expressed as soluble proteins in a T7 bacterial expression system. Both proteins are independently active nucleotide diphosphate kinases and readily form intra- and intermolecular disulfide bonds. The biochemical properties of these proteins may explain the diversity of mature eucaryotic nucleoside diphosphate kinases.

Animals↗
PubMed1992

Insulin stimulation of phosphatidylinositol 3-kinase activity and association with insulin receptor substrate 1 in liver and muscle of the intact rat.

Growth factors stimulate the enzyme phosphatidylinositol (PI) 3-kinase in cells in culture. Insulin rapidly stimulates tyrosine phosphorylation of its endogenous substrate, insulin receptor substrate 1 (IRS-1), and in vitro IRS-1 associates with PI 3-kinase, thus activating the enzyme. We have examined whether insulin is capable of stimulating the PI 3-kinase pathway in two physiological target tissues for the actions of insulin in vivo, liver and skeletal muscle. After intraportal injection of insulin into anesthetized rats, there was a 2-fold stimulation of total hepatic PI 3-kinase activity in liver and muscle extracts and a 10- to 20-fold increase in PI 3-kinase activity immunoprecipitated with anti-IRS-1 antibodies. Stimulation of PI 3-kinase was accompanied by an association between this enzyme and IRS-1 as detected by immunoprecipitation of liver and muscle extracts with anti-IRS-1 antibodies and Western blotting with antibodies to the 85-kDa subunit of PI 3-kinase. Immunoprecipitation with anti-p85 antibodies and phosphotyrosine immunoblotting revealed no tyrosine phosphorylation of PI 3-kinase, but demonstrated co-precipitation of tyrosine-phosphorylated IRS-1, as well as another phosphotyrosine protein of approximately 135-140 kDa. Thus, IRS-1 phosphorylation plays a significant role in the activation of PI 3-kinase in vivo by insulin.

Animals↗
PubMed1992

Expression and function of IRS-1 in insulin signal transmission.

IRS-1 is a major insulin receptor substrate which may play an important role in insulin signal transmission. The mRNA for IRS-1 in rat cells and tissues is about 9.5 kilobases (kb). Rat liver IRS-1 was stably expressed in Chinese hamster ovary (CHO) cells (CHO/IRS-1). Although its calculated molecular mass is 131 kDa, IRS-1 from quiescent cells migrated between 165 and 170 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRS-1 was phosphorylated strongly on serine residues and weakly on threonine residues before insulin stimulation. Insulin immediately stimulated tyrosine phosphorylation of IRS-1, and after 10-30 min with insulin its apparent molecular mass increased to 175-180 kDa. Expression of the human insulin receptor and rat IRS-1 together in CHO/IR/IRS-1 cells increased the basal serine phosphorylation of IRS-1 and strongly increased tyrosine phosphorylation during insulin stimulation. Purified insulin receptors directly phosphorylated baculovirus-produced IRS-1 exclusively on tyrosine residues. By immunofluorescence, IRS-1 was absent from the nucleus, but otherwise distributed uniformly before and after insulin stimulation. Some IRS-1 associated with the insulin receptor during insulin stimulation. In addition, a phosphatidylinositol 3'-kinase associated with IRS-1 during insulin stimulation, and this association was more sensitive to insulin in CHO cells overexpressing the insulin receptor (CHO/IR cells), more responsive to insulin to CHO/IRS-1 cells, and both sensitive and responsive in CHO/IR/IRS-1 cells. Similarly, insulin-stimulated DNA synthesis was more sensitive to insulin in CHO/IR cells, and more responsive in CHO/IRS-1 cells; however, insulin-stimulated DNA synthesis was sensitive but poorly responsive to insulin in CHO/IR/IRS-1 cells. Together, these results suggest that IRS-1 is a direct physiologic substrate of the insulin receptor and may play an important role in insulin signal transmission.

Amino Acid Sequence↗
PubMed1992

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.

3T3 Cells↗
PubMed1992

Insulin differentially regulates protein phosphotyrosine phosphatase activity in rat hepatoma cells.

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.

Amino Acid Sequence↗
PubMed1992

The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies.

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.

Animals↗
PubMed1992

Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation.

We have studied the phosphatidylinositol 3-kinase (PtdIns 3-kinase) in insulin-stimulated Chinese hamster ovary (CHO) cells expressing normal (CHO/IR) and mutant human insulin receptors. Insulin stimulation of CHO/IR cells results in an increase in PtdIns 3-kinase activity associated with anti-phosphotyrosine (alpha PY) immunoprecipitates, which has been previously shown to correlate with the in vivo production of PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (Ruderman, N., Kapeller, R., White, M.F., and Cantley, L.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). Stimulation was maximal within 1 min and showed a dose response identical to that of insulin receptor autophosphorylation. The PtdIns 3-kinase also associated with the insulin receptor in an insulin-stimulated manner, as approximately 50% of the total alpha PY-precipitable activity could be specifically immunoprecipitated with anti-insulin receptor antibody. Mutant insulin receptors displayed variable ability to stimulate the PtdIns 3-kinase, but in all cases the presence of PtdIns 3-kinase in alpha PY immunoprecipitates correlated closely with the tyrosyl phosphorylation of the endogenous substrate pp185. In CHO cells expressing a kinase-deficient mutant (IRA1018), there was no observable insulin stimulation of PtdIns 3-kinase activity in alpha PY immunoprecipitates and no tyrosyl phosphorylation of pp185. Substitution of Tyr1146 in the insulin receptor regulatory region with phenylalanine partially impaired receptor autophosphorylation, pp185 phosphorylation, and insulin-stimulated increases in alpha PY-precipitable PtdIns 3-kinase activity. In contrast, a deletion mutant lacking 12 amino acids from the juxtamembrane region (IR delta 960) displayed normal in vivo autophosphorylation but failed to stimulate the PtdIns 3-kinase or phosphorylate pp185. Finally, a mutant receptor from which the C-terminal 43 amino acids had been deleted (IR delta CT) exhibited normal insulin-stimulated autophosphorylation, pp185 phosphorylation, and stimulation of the PtdIns 3-kinase activity in alpha PY immunoprecipitates. These data suggest that the PtdIns 3-kinase is itself a substrate of the insulin receptor kinase or associates preferentially with a substrate. A comparison of the biological activities of the mutant receptors with their activation of the PtdIns 3-kinase furthermore suggests that the PtdIns 3-kinase may be linked to insulin's ability to regulate DNA synthesis and cell growth.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation.

IRS-1 undergoes rapid tyrosine phosphorylation during insulin stimulation and forms a stable complex containing the 85 kDa subunit (p85) of the phosphatidylinositol (PtdIns) 3'-kinase, but p85 is not tyrosyl phosphorylated. IRS-1 contains nine tyrosine phosphorylation sites in YXXM (Tyr-Xxx-Xxx-Met) motifs. Formation of the IRS-1-PtdIns 3'-kinase complex in vitro is inhibited by synthetic peptides containing phosphorylated YXXM motifs, suggesting that the binding of PtdIns 3'-kinase to IRS-1 is mediated through the SH2 (src homology-2) domains of p85. Furthermore, overexpression of IRS-1 potentiates the activation of PtdIns 3-kinase in insulin-stimulated cells, and tyrosyl phosphorylated IRS-1 or peptides containing phosphorylated YXXM motifs activate PtdIns 3'-kinase in vitro. We conclude that the binding of tyrosyl phosphorylated IRS-1 to the SH2 domains of p85 is the critical step that activates PtdIns 3'-kinase during insulin stimulation.

Amino Acid Sequence↗
PubMed1992

The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals.

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.

Amino Acid Sequence↗
PubMed1991

Effects of farnesylcysteine analogs on protein carboxyl methylation and signal transduction.

Several proteins associated with signal transduction in eukaryotes are carboxyl methylated at COOH-terminal S-farnesylcysteine residues. These include members of the Ras superfamily and gamma-subunits of heterotrimeric G-proteins. The enzymes that catalyze the carboxyl methylation reaction also methylate small molecules such as N-acetyl-S-trans, trans-farnesyl-L-cysteine (AFC). AFC inhibits carboxyl methylation of p21ras and related proteins both in vitro and in vivo. Saturating concentrations of AFC cause a greater than 80% inhibition of chemotactic responses of mouse peritoneal macrophages. Our results suggest that carboxyl methylation may play a role in the regulation of receptor-mediated signal transduction processes in eukaryotic cells.

Alkyl and Aryl Transferases↗