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J E Porter

Publications and source records attributed to J E Porter.

At least 19 recordsLinked to original sources
PubMed2000

Influence of a lysine 331 counterion on the pK(a) of aspartic acid 125: evidence for a salt-bridge interaction and role in alpha(1b)-adrenergic receptor activation.

We have hypothesized previously that a salt-bridge constraint exists in the alpha(1b)-adrenergic receptor (AR). Docking of the agonist epinephrine can disrupt this constraint via competition of its protonated amine, leading to an agonist-induced activation of second messengers. The amino acids, K331 and D125, which comprise this salt-bridge, should be closely associated with each other in the unbound form of the alpha(1b)-AR. This ionic association should stabilize the negative charge of D125, leading to an increase in its acid strength or a decreased pK(a). If the charged state of D125 is important for agonist binding, then changing the type of amino acid at position 331 should decrease the acid strength of D125, leading to epinephrine affinity changes for the alpha(1b)-AR. To test this hypothesis, site-directed mutagenesis was performed at position 331 of the alpha(1b)-AR. The effect these substitutions had on D125 acid strength was quantitated via epinephrine affinity changes calculated from competition binding experiments performed at different pH values. For all mutations of the alpha(1b)-AR where the positive charge at position 331 was eliminated, there was a significant increase in the pK(a) ( congruent with 0.73) of an acidic amino acid(s). In addition, there was an increase in the binding affinity of epinephrine for these mutants that was associated with a gain in the basal production of inositol triphosphates. These results are consistent with an aspartic acid residue as the counterion for K331 of the salt-bridge constraint, which disrupted, is a part of the receptor activation process. Moreover, changes in the pK(a) of D125 were not dependent on the type of amino acid substituted at position 331. This suggests a mechanism in which K331 is no longer influencing D125 after salt-bridge disruption in the wild-type alpha(1b)-AR, but may move to another stabilized position, analogous to what has been suggested for bacteriorhodopsin. Differences from the wild-type receptor in D125 pK(a) for the K331 mutations were used to estimate the free-energy potential of the constraining salt-bridge. This free energy ( congruent with 1 kcal/mol) is significant, but weak enough to be consistent with an activational mechanism where docking of the receptor agonist has sufficient free energy to cause disruption of the salt-bridge.

Animals↗
PubMed1999

Characteristics for a salt-bridge switch mutation of the alpha(1b) adrenergic receptor. Altered pharmacology and rescue of constitutive activity.

Agonist-dependent activation of the alpha(1)-adrenergic receptor is postulated to be initiated by disruption of an interhelical salt-bridge constraint between an aspartic acid (Asp-125) and a lysine residue (Lys-331) in transmembrane domains three and seven, respectively. Single point mutations that disrupt the charges of either of these residues results in constitutive activity. To validate this hypothesis, we used site-directed mutagenesis to switch the position of these amino acids to observe, if possible, regeneration of the salt-bridge reverses that the constitutive activity of the single point mutations. The transiently expressed switch mutant receptor displayed an altered pharmacological profile. The affinity of selective alpha(1b)-adrenergic receptor antagonists for the switch mutant (D125K/K331D) was no different from the wild-type alpha(1b)-adrenergic receptor, suggesting that both receptors are maintaining similar tertiary structures in the cell membrane. However, there was a significant 4-6-fold decrease in the affinity of protonated amine receptor agonists and a 3-6-fold increase in the affinity of carboxylated catechol derivatives for the switch mutant compared with the wild-type alpha(1b)-adrenergic receptor. This pharmacology is consistent with a reversed charge at position 125 in transmembrane domain three. Interestingly, the ability of either a negatively or positively charged agonist to generate soluble inositol phosphates was similar for both types of receptors. Finally, the switch mutant (D125K/K331D) displayed similar basal signaling activity as the wild-type receptor, reversing the constitutive activity of the single point mutations (D125K and K331D). This suggests an ionic constraint has been reformed in the switch mutant analogous to the restraint previously described for the wild-type alpha(1b)-adrenergic receptor. These results strongly establish the disruption of an electrostatic interaction as an initial step in the agonist-dependent activation of alpha(1)-adrenergic receptors.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1998

Identification of a conserved switch residue responsible for selective constitutive activation of the beta2-adrenergic receptor.

A cysteine-to-phenylalanine mutation of residue 116 in the third transmembrane domain of the beta2-adrenergic receptor caused selective constitutive activation of Na+/H+ exchange through a pathway not involving cAMP. This selectivity was identified by comparing binding and signaling characteristics of wild-type (WT) versus C116F mutant receptors transiently transfected into COS-1 cells. Indicating constitutive activity, ligand binding to the C116F mutant showed a 78-fold higher than WT affinity for isoproterenol and a 40-fold lower than WT affinity for ICI 118551. Although agonist-independent activation of cAMP production was not exhibited by the C116F mutant, a constitutive stimulation of the Na+/H+ exchanger (NHE1) was observed. This was identified by measuring either basal intracellular pH (pHi) or rate of pHi recovery from cellular acid load. Due to a higher rate of H+ efflux through NHE1, C116F transfectants exhibited a significantly higher pHi (7.42) than did WT transfectants (7.1). Furthermore, the rate of pHi recovery from acid load facilitated by NHE1 was 2.1-fold faster in mutant transfectants than in WT transfectants. The lower rate seen in the WT case was stimulated by epinephrine, and the higher rate seen in the mutant case was inhibited by ICI 118551. These findings, which show that a C116F mutation of the beta2-adrenergic receptor evokes selective constitutive coupling to NHE1 over cAMP, form the basis of our prediction that multiple and distinct activation states can exist in G protein-coupled receptors.

Adrenergic beta-Agonists↗
PubMed1998

The agonism and synergistic potentiation of weak partial agonists by triethylamine in alpha 1-adrenergic receptor activation: evidence for a salt bridge as the initiating process.

Alpha 1-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha 1-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha 1b-AR and in Rat-1 fibroblasts stably transfected with the human alpha 1a-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha 1-AR agonists and this effect was fully inhibited by the alpha 1-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha 1-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha 1-ARs with a Ki of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha 1-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha 1-AR activation.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1997

Positional isomers of monopegylated interferon alpha-2a: isolation, characterization, and biological activity.

The success of recombinant interferon alpha in the clinic in part is limited by two properties of the protein: short serum half-life and immunogenicity. To improve these properties, interferon alpha-2a was conjugated with polyethylene glycol (PEG-5000). A homogeneous preparation of monopegylated interferon alpha-2a was subjected to vigorous analytical and activity characterization. A newly developed ampholyte-free chromatofocussing-like cation-exchange HPLC method utilizing a sulfopropyl resin was used to separate the monopegylated protein into 11 species. Peptide mapping, sequencing, and mass spectrometric analyses indicated that these species are positional isomers where each isomer represents a single polymer molecule conjugated to one specific lysine residue. No species with a modification at the amino terminus was observed. All 11 isomers show antiviral and antiproliferative activities in the same range as the parent monopegylated interferon alpha-2a.

Animals↗
PubMed1997

Synergism of constitutive activity in alpha 1-adrenergic receptor activation.

Recently a number of mutations have been found in vitro which maintain alpha 1-adrenergic receptors (ARs) in a partially activated form. We have previously identified two amino acid residue positions in the alpha 1b-adrenergic receptor (AR), Cys128 and Ala204, one in each of the third and fifth transmembrane segments, that constitutively activate the receptor when substituted for a phenylalanine or valine, respectively [Perez et al. (1996) Mol. Pharmacol. 49, 112-122; Hwa et al. (1996) J. Biol. Chem. 271, 7956-7964]. Another mutation analyzed previously, Ala293Glu, located in the third intracellular loop, also constitutively activates the receptor [Kjelsborg et al. (1992) J. Biol. Chem. 267, 1430-1433]. All three mutations displayed similar manifestations of constitutive activity such as higher binding affinity and potency for agonists as well as higher basal signal transduction as predicted by the revised ternary complex model of receptor activation. We hypothesized that the individual mutations because of their critical location alter the conformation of the transmembrane helices such that mimicry occur that partially conforms to the activated state, R*. To explore whether these potential conformations are independent, we combined these three mutations in all possible permutations. The combined triple mutation displays 700-fold higher binding affinity for (-)-epinephrine and 20-fold higher basal IP3 release than the wild-type receptor. We also observed that each mutation contributed independently and synergistically to both receptor agonist binding and activation with the combined mutations basal activity exceeding that of the fully-stimulated wild-type receptor. There was also a direct correlation between epinephrine's binding affinity and the degree of constitutive activity. Because the mutations affect different transmembrane domains, these results are consistent with a mechanism that helical movement acts in a concerted fashion in agonist-induced activation, a synergism predicted if multiple helix movement is involved in receptor activation.

Animals↗
PubMed1996

Activation of the alpha1b-adrenergic receptor is initiated by disruption of an interhelical salt bridge constraint.

Rhodopsin receptor activation involves the disruption of a salt bridge constraint between glutamic acid 113 on transmembrane 3 and a lysine 296 in transmembrane 7, which forms a Schiff's base with retinal. Light-induced isomerization of cis-retinal to the all trans form breaks this rhodopsin salt bridge leading to receptor activation. The analogous residues in alpha1b-adrenergic receptors, aspartic acid 125 and lysine 331, also have the potential of forming a constraining salt bridge holding the receptor to an inactive protein configuration. This alpha1b-adrenergic receptor salt bridge constraint is then released upon binding by the receptor agonist. To test this hypothesis, site-directed mutagenesis was used to eliminate the positive charge at position 331 by substitution of an alanine. The expressed alpha1b-adrenergic receptor mutant demonstrated a 6-fold increased epinephrine binding affinity with no alterations of affinity values for selective adrenergic receptor antagonists. Furthermore, an increased epinephrine potency for total soluble inositol phosphate production along with an elevated basal inositol triphosphate level was observed in COS-1 cells transfected with mutant versus wild-type alpha1b-adrenergic receptors. Similar results were obtained for a lysine to a glutamic acid alpha1b-adrenergic receptor mutation. In addition, increased basal inositol triphosphate levels were also observed for two aspartic acid 125 alpha1b-adrenergic receptor mutations, consistent with this residue's role as the counterion of the salt bridge. Taken together, these alpha1b-adrenergic receptor mutations suggest a molecular mechanism by which the positively charged lysine 331 stabilizes the negatively charged aspartic acid 125 via a salt bridge constraint until bound by the receptor agonist.

Animals↗
PubMed1995

The Benchmarking Effort for Networking Children's Hospitals (BENCHmark).

BACKGROUND: In 1992, 12 large children's hospitals established the Benchmarking Effort for Networking Children's Hospitals (BENCHmark). The goal was for the BENCHmark effort to supplement the hospitals' continuous quality improvement (CQI) programs and to speed adoption of best practices from peer institutions. For three years, the hospitals have been comparing data on cost, quality, and speed indicators. Also, "best practice" groups have met to share information on how processes can be improved. RESULTS: The BENCHmark hospitals have experienced significant process improvement in areas such as emergency department waiting time and admitting process time. EXAMPLE: The BENCHmark hospitals selected admitting as one of the first best practice groups to meet. Interdisciplinary staff from all BENCHmark hospitals met three times over the course of a year to define their indicator and share information on best practices. St Louis Children's Hospital, as a result, instituted a pre-arrival team and cross-trained staff, with the result being a reduction of admitting processing time from 58 minutes to 19 minutes. Same-day surgery patients now bypass the admitting department and go directly to the surgical floor. Patient and surgeon satisfaction has increased greatly. CONCLUSIONS: Hospitals that are planning to benchmark are encouraged to reach consensus on project goals and to focus on indicators that provide a clear business advantage. Physician involvement is key to improving performance and physicians will only be engaged if the hospitals against whom they are benchmarked are considered peers. Being willing to share initial data openly seems to be a key factor in determining successful integration of the BENCHmark process into hospital CQI efforts. The BENCHmark project has been so successful that a second group of 12 comparable pediatric institutions, known as the Network II, has been established.

Efficiency, Organizational↗
PubMed1994

Differential actions of lamprey peptide methionine-tyrosine at Y1 and Y2 neuropeptide Y receptors.

Peptide methionine-tyrosine (PMY), a peptide of the neuropeptide Y (NPY) superfamily isolated from the brain and intestine of the sea lamprey, had the same maximum effect but was 11-fold less potent than pig NPY in inhibiting field-stimulated contraction of the rat vas deferens, an effect mediated through the Y2 receptor. In contrast, PMY produced a 9-fold greater maximum effect but was 3-fold less potent than pig NPY in contracting the guinea pig mesenteric artery, an effect mediated through the Y1 receptor. Molecular modelling has suggested that the conformation of PMY is appreciably different from NPY only in the beta-turn region of the molecule (residues 9-14). Our data suggest, therefore, that modifications in this region of NPY may useful in the design of receptor selective analogs.

Amino Acid Sequence↗
PubMed1994

Purpose in life and outcome of treatment for alcohol dependence.

A number of studies have linked the development of substance abuse problems to a lack of purpose or meaning in life, and a few studies have demonstrated an increase in sense of life purpose through substance abuse treatment programmes. The present study extended past research by examining the relationship of purpose in life to treatment outcome assessed three months after completion of treatment. The subject sample comprised 131 people in in-patient treatment programmes or awaiting treatment for alcoholism (in some cases in addition to other drug addictions). Consistent with previous research, the mean Purpose in Life Test (PIL) score before treatment was significantly below the normal range and the mean PIL score at the end of in-patient treatment was within the normal range. Furthermore, the PIL score at the end of treatment was predictive of changes in intimate relationships and health at follow-up. It was also predictive of follow-up drinking/drug use status. However, the pattern of prediction differed in the two treatment groups. Post-treatment PIL score was a positive predictor of improvement in a skill-based treatment centre, and a negative predictor in a more authoritarian, confrontation-based programme. The distinction between internally and externally derived senses of meaning is presented as one possible explanation of these findings.

Adaptation, Psychological↗
PubMed1993

Nurse triage.

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Emergency Nursing↗