Biomedical subjects

K Nandagopal

Publications and source records attributed to K Nandagopal.

6 recordsLinked to original sources

PubMed2001

Critical role for nitric oxide signaling in cardiac and neuronal ischemic preconditioning and tolerance.

Preconditioning to ischemic tolerance is a phenomenon in which brief episodes of a subtoxic insult induce a robust protection against the deleterious effects of subsequent, prolonged, lethal ischemia. The subtoxic stimuli that constitute the preconditioning event are quite diverse, ranging from brief ischemic episodes, spreading depression or potassium depolarization, chemical inhibition of oxidative phosphorylation, exposure to excitotoxins and cytokines. The beneficial effects of preconditioning were first demonstrated in the heart; it is now clear that preconditioning can induce ischemic tolerance in a variety of organ systems including brain, heart, liver, small intestine, skeletal muscle, kidney, and lung. There are two temporally and mechanistically distinct types of protection afforded by preconditioning stimuli, acute and delayed preconditioning. The signaling cascades that initiate the acute and delayed preconditioning responses may have similar biochemical components. However, the protective effects of acute preconditioning are protein synthesis-independent, mediated by post-translational protein modifications, and are short-lived. The effects of delayed preconditioning require new protein synthesis and are sustained for days to weeks. Elucidation of the molecular mechanisms that are involved in preconditioning and ischemic tolerance and identification of drugs that mimic this protective response have the potential to improve the prognosis of patients at risk for ischemic injury. This article focuses on recent findings on the effects of ischemic preconditioning in the cardiac and nervous systems and discusses potential targets for a successful therapeutic approach to limit ischemia-reperfusion injury.

Animals↗

PubMed1999

Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions.

Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis. When assayed for receptor tyrosine kinase stimulation using (Glu4,Tyr1)n as the exogenous substrate in vitro, the relative agonist activities of position 16 mutants range from 14-263% of wild-type hEGF. The rank order of potency was found to correlate with the relative receptor binding affinities of the mutants, which range from 7-272% of wild-type, as determined by radioreceptor competition assays. The mitogenic activity of the H16 mutants is similar to that of wild-type hEGF as determined by clonogenic assays using rat tracheal epithelial cells. While the colony forming efficiencies do not reflect significant differences in growth rate or survival characteristics in the presence of the hEGF variants, it is reduced to 1.6% in control cultures which lack EGF in the medium. The results show that H16 of hEGF, although not essential for mitogenic activity, optimizes receptor recognition by hydrogen-bond donor/acceptor interactions and may share this feature with H18 of hTGFalpha.

Animals↗

PubMed1998

Structure-function studies of ligand-induced epidermal growth factor receptor dimerization.

We present a novel 96-well assay which we have applied to a structure-function study of epidermal growth factor receptor dimerization. The basis of the assay lies in the increased probability of EGFRs being captured as dimers by a bivalent antibody when they are immobilized in the presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultant 5-7-fold increase in binding of a radiolabeled ligand probe. When the assay was applied to members of the EGF ligand family, murine EGF, transforming growth factor alpha, and heparin-binding EGF-like growth factor were comparable with human EGF (EC50 = 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR1-84), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 microM. We further probed the involvement of the C-tail and the conserved leucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGFalpha44-50 and the EGF point mutant L47A) and a C-terminally extended form of AR (AR1-90) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGFalpha44-50 > L47A = L15A >> AR1-90 > AR1-84. Although AR was much worse than predicted from its affinity, this defect could be partially rectified by co-localization of the immobilizing antibody with heparin. Thus, it seems likely that AR cannot dimerize the EGFR unless other accessory molecules are present to stabilize its functional association with the EGFR.

Cell Line↗

PubMed1996

The EGF receptor binding of recombinant heregulinbeta1/EGF hybrids is blocked by heregulin residue glutamate 195.

Defined sequences from the EGF-like domain of human heregulin-beta1 (HRGbeta1) were recombined with a synthetic gene for human epidermal growth factor (hEGF) in an attempt to locate receptor-specific determinants within the HRGbeta1 molecule that blocks its inappropriate association with the EGF receptor (EGFR). Receptor competition assays detected only minor changes in relative EGFR affinity for those hybrids containing up to 12 N-terminal HRGbeta1 residues. However, extending the N-terminal substitution to include 20 HRGbeta1 residues resulted in a 100-fold drop in relative EGFR binding. Both interruption of the major beta-sheet structure of hEGF by insertion of a three amino acid loop present in HRGbeta1 and replacement of nearly the entire C-terminal hEGF subdomain with segments of HRGbeta1 sequence resulted in a 5-fold decreased EGFR affinity. The results presented here demonstrate that while a substantial portion of the hEGF and HRGbeta1 protein sequences were nearly interchangeable with regard to EGFR binding, the introduction of HRGbeta1 residue Glu195 effected a major decrease in EGFR binding.

Amino Acid Sequence↗

PubMed1996

The functional importance of Leu15 of human epidermal growth factor in receptor binding and activation.

The biological importance of Leu15 of epidermal growth factor (EGF) is suggested by its conservation through evolution, its critical location in the domain-domain interface of EGF and its close proximity to Arg41, a residue that is crucial for receptor binding and activation. Mutagenesis of Leu15 of human EGF (hEGF) was employed to examine the role of this residue in the ligand-receptor interaction. The relative receptor affinities of the hEGF variants, as determined by radioreceptor competition assays, varied depending on the amino acid substitution. The L15F, L15W and L15V hEGF analogues had receptor affinities 45, 26 and 18% respectively of wild type hEGF. The L15A and L15R analogues displayed receptor affinities of only 2.4 and 1.6% relative to wild type hEGF. No binding of the L15E analogue was detected. The relative agonist activities, as measured by receptor tyrosine kinase stimulation assays, generally followed a similar trend. The L15F, L15W and L15V analogues stimulated the receptor kinase to a level (Vmax) similar to that for wild type hEGF. A striking difference was observed between the L15A and L15R variants; although having similar binding affinities, the L15A mutant activated the receptor to only approximately 5% of the wild type Vmax in contrast to 53% for the L15R mutant. 1H-NMR analysis of the L15R and L15A mutants showed only minor structural alterations that were not sufficient to account for the dramatic losses in binding and agonist activities. The results indicate that both the size and hydrophobicity of the gamma-branched aliphatic side chain of Leu15 of hEGF are important in the formation of a catalytically active ligand-receptor complex.

Base Sequence↗

PubMedDate not supplied

Utilization of a receptor reserve for effective amplification of mitogenic signaling by an epidermal growth factor mutant deficient in receptor activation.

The idea of a receptor reserve in mediating cellular function is well known but direct biochemical evidence has not been easy to obtain. This study stems from our results showing that L15 of epidermal growth factor (EGF) is important in both EGF receptor (EGFR) binding and activation, and the L15A analog of human EGF (hEGF) partially uncouples EGFR binding from EGFR activation (Nandagopal et al., [1996] Protein Engng 9:781-788). We address the cellular mechanism of mitogenic signal amplification by EGFR tyrosine kinase in response to L15A hEGF. L15A is partially impaired in receptor dimerization, shown by chemical cross-linking and allosteric activation of EGFR in a substrate phosphorylation assay. Immunoprecipitation experiments reveal, however, that L15A can induce EGFR autophosphorylation in intact murine keratinocytes by utilizing spare receptors, the ratio of total phosphotyrosine content per receptor being significantly lower than that elicited by wild-type. This direct biochemical evidence, based on function, of utilization of a receptor reserve for kinase stimulation suggests that an EGF variant can activate varying receptor numbers to generate the same effective response. L15A-activated receptors can stimulate mitogen-activated protein kinase (MAPK) that is important for mitogenesis. The lack of linear correlation between levels of receptor dimerization, autophosphorylation, and MAPK activation suggests that signal amplification is mediated by cooperative effects. Flow cytometric analyses show that the percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S-phase are both decreased relative to 1 nM wild-type, indicating that MAPK activation alone is insufficient for maximal stimulation of mitogenesis. Higher concentrations of L15A reverse this effect, indicating that L15A and wild-type differ in the number of receptors each activates to induce the threshold response, which may be attained by cooperative activation of receptor dimers/oligomers by van der Waal's weak forces of attraction. The maintenance of a receptor reserve underscores an effective strategy in cell survival.

Alanine↗