G protein-coupled receptors for dopamine and serotonin control signaling pathways targeted by many psychoactive drugs. A puzzle is how receptors with similar functions and nearly identical binding site structures, such as D2 dopamine receptors and 5-HT2A serotonin receptors, could evolve a mechanism that discriminates stringently in their cellular responses between endogenous neurotransmitters. We used the Difference Evolutionary Trace (Difference-ET) and residue-swapping to uncover two distinct sets of specificity-determining sequence positions. One at the ligand-binding pocket determines the relative affinities for these two ligands, and a distinct, surprising set of positions outside the binding site determines whether a bound ligand can trigger the conformational rearrangement leading to G protein activation. Thus one site specifies affinity while the other encodes a filter for efficacy. These findings demonstrate that allosteric pathways linking distant interactions via alternate conformational states enforce specificity independently of the ligand-binding site, such that either one may be rationally rekeyed to different ligands. The conversion of a dopamine receptor effectively into a serotonin receptor illustrates the plasticity of GPCR signaling during evolution, or in pathological states, and suggests new approaches to drug discovery, targeting both classes of sites.

Objective To determine the predictive value of discharge destination as a surrogate for defining unfavorable outcome at 3- and 12-months poststroke. Design Analysis of the prospectively collected data from a randomized, placebo-controlled trial in patients with ischemic stroke presenting within 3 hours of symptom onset. Setting Post hoc analysis of patients recruited in a clinical trial. Participants Patients (N=530) discharged alive from the hospital after ischemic stroke. Interventions Not applicable. Main Outcome Measures Positive and negative predictive value and likelihood ratios of discharge destination for unfavorable outcome at 3- and 12-months poststroke defined by a Modified Rankin Scale (MRS) score of 2 to 6, 3 to 6, or 4 to 6. A likelihood ratio indicates how many times more (or less) likely a particular discharge destination is seen in patients with an unfavorable outcome compared with those without unfavorable outcome. Results The positive predictive value of nursing home and rehabilitation facility discharges was highest for unfavorable outcome defined by an MRS score of 2 to 6 (95%) and rehabilitation facility (89%) at 3-months poststroke, respectively. The positive predictive value of rehabilitation facility/nursing home (90%) was also highest for unfavorable outcomes defined by an MRS score of 2 to 6 compared with those defined by MRS scores of 3 to 6 (79%) and 4 to 6 (57%). The positive likelihood ratio was highest for nursing home discharges (13; 95% confidence interval [CI], 4.1– 41) followed by rehabilitation facility discharges for unfavorable outcome defined by an MRS score of 2 to 6 at 3-months poststroke (5.3; 95% CI, 3.5–7.9). The negative likelihood ratio was the highest for home discharge for unfavorable outcome defined by an MRS score of 2 to 6 (4.5; 95% CI, 3.4 – 6.1). A similar pattern was observed with unfavorable outcome defined using various thresholds at 12 months. Conclusions Discharge destination can provide high predictive values and likelihood ratios for death and disability at 3-months poststroke, as defined by an MRS of score of 2 to 6.

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Elderly patients presenting with acute ischemic stroke or transient ischemic attack have high plasma osmolality levels, suggestive of volume depletion. This seems to be an early phenomenon and possibly a contributing factor to cerebral ischemia.

To determine the outcomes related to thrombolytic treatment of an acute ischemic stroke secondary to an arterial dissection in a large national cohort.

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Background: Subclinical cancer can manifest as a thromboembolic event and may be detected at a later interval in ischemic stroke survivors. We determined the rate of incident cancer and effect on cardiovascular endpoints in a large cohort of ischemic stroke survivors. Methods: An analysis of 3,680 adults with nondisabling cerebral infarction who were followed for two years within the randomized, double-blinded VISP trial was performed. The primary intervention was best medical/surgical management plus a daily supplementation of vitamin B6, vitamin B12, and folic acid. We calculated age-adjusted rates of incidence of cancer among ischemic stroke survivors and standardized incidence ratios (SIR) with 95% confidence intervals (CI) based on comparison with age-adjusted rates in the general population. The significant variables from univariate analysis were entered in a Cox Proportional Hazards analysis to identify the association between various baseline factors and incident cancer after adjusting age, gender, and race/ethnicity. A logistic regression analysis evaluated the association between incident cancer and various endpoints including stroke, coronary heart disease, myocardial infarction, and death after adjusting age, gender, and race/ethnicity. Results: A total of 3,247 patients (mean age ± SD of 66 ± 11; 2,013 were men) were cancer free at the time of enrollment. The incidence of new cancer was 0.15, 0.80, 1.2, and 2.0 per 100 patients at 1 month, 6 months, 1 year, and 2 years, respectively. The age-adjusted annual rate of cancer in patients with ischemic stroke was higher than in persons in the general population at 1 year (581.8/100,000 persons vs. 486.5/100,000 persons, SIR 1.2, 95% CI 1.16-1.24) and 2 years (1,301.7/100,000 vs. 911.5/100,000, SIR 1.4, 95% CI 1.2-1.6) after recruitment. There was a higher risk for death (odds ratio (OR) 3.1, 95% CI 1.8-5.4), and composite endpoint of stroke, coronary heart disease, and/or death (OR 1.4, 95% CI 1.0-2.2) among participants who developed incident cancer compared with those who were cancer free after adjusting for potential confounders. Conclusions: The annual rate of age-adjusted cancer incidence was higher among ischemic stroke patients compared with those in the general population. The odds of mortality were three folds higher among stroke survivors who developed incident cancer.

The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues.allostery | G protein-coupled receptors | residue covariation | Evolutionary Trace I dentifying residues that coevolved to maintain or acquire fitness properties is critical for understanding protein structure, function, and evolution (1). Previous studies have shown that covarying residue pairs, those that exhibit correlated amino acid changes in large multiple sequence alignments, tend to form structural contacts (2-7), enhancing predictions of protein 3D structures (8-11). Covariation can also involve distal residues, but the function of these at-a-distance couplings is elusive and has been attributed to background noise, alternative protein conformations, or subunit interactions of protein homooligomers (5,7,12). Alternately, distal covarying residue pairs could indicate allosteric couplings (6,(13)(14)(15)(16)(17)(18).The possibility of capturing intramolecular allosteric communication by amino acid covariation analysis of protein family sequences has not been extensively explored. Nonproximal thermodynamic coupling between correlated residue pairs was noted in 274 PDZ domains (14), but the relationship to allostery is still debated (19,20). It may be that distinctive allosteric mechanisms, even among close homologs, limit the extraction of allosteric couplings from ...