Agonist-stimulated desensitization of the ␤ 2 -adrenergic receptor (␤ 2 AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to ␤-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human ␤ 2 AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant ␤ 2 ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E 1 , and forskolin at maximum concentrations stimulated phosphorylation of the ␤ 2 AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC 50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC 50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC 50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still Ϸ200-fold greater than for the PKA site. The EC 50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the ␤ 2 AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agoniststimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine Ն formoterol ϭ fenoterol Ͼ terbutaline ϭ zinterol ϭ albuterol Ͼ salmeterol Ͼ Ͼ dobutamine Ն ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous ␤ 2 AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.The ␤ 2 -adrenergic receptor (␤ 2 AR) plays significant roles in relaying signals from the autonomic sympathetic nervous system to the cardiovascular and pulmonary systems in particular. There are...

The human beta(2)-adrenergic receptor (betaAR) is rapidly desensitized in response to saturating concentrations of agonist by G protein-coupled receptor kinases (GRKs) and cAMP-dependent protein kinase A (PKA) phosphorylation of the betaAR, followed by beta-arrestin binding and receptor internalization. betaAR sites phosphorylated by GRK in vivo have not yet been identified. In this study, we examined the role of the carboxyl terminal serines, 355, 356, and 364, in the GRK-mediated desensitization of the betaAR. Substitution mutants of these serine residues were constructed in which either all three (S355,356,364A), two (S355,356A and S356, 364A), or one of the serines (S356A and S364A) were modified. These mutants were constructed in a betaAR in which the serines of the PKA consensus site were substituted with alanines (designated PKA(-)) to eliminate any PKA contribution to desensitization, and they were stably transfected into human embryonic kidney 293 cells. Treatment of the PKA(-) mutant with 10 microM epinephrine for 5 min caused a 3. 5-fold increase in the EC(50) value and a 42% decrease in the V(max) value for epinephrine stimulation of adenylyl cyclase. Substitution of all three serines completely inhibited the epinephrine-induced shift in the EC(50). Both double mutants, S355,356A and S356,364A, showed a nearly complete loss of the EC(50) shift, whereas the single substitutions, S356A and S364A, caused only a slight decrease in desensitization. None of the mutations altered the epinephrine-induced decrease in V(max,) which seems to be downstream of the receptor. The triple mutation caused a 45% decrease in epinephrine-induced internalization and a 90 to 95% reduction in phosphorylation of the betaAR relative to the PKA(-) (1.9+/- 0.2- and 16.6+/-3.8-fold phosphorylation over basal, respectively). The double mutants caused an intermediate reduction in internalization (20-21%) and phosphorylation (43-52%). None of the serine mutations altered the rate of betaAR recycling. Our data demonstrate that the cluster of serines within the 355 to 364 betaAR domain confer the rapid, GRK-mediated, receptor-level desensitization of the betaAR.

BACKGROUND No prospective cohort study of high-risk children has used rigorous exposure assessment and optimal diagnostic procedures to examine the perinatal antecedents of autism spectrum disorder (ASD), separately among those with and without cognitive impairment. OBJECTIVE To identify perinatal factors associated with increased risk for ASD with and without intellectual disability (ID: IQ < 70) in children born extremely preterm. STUDY DESIGN This prospective multi-center (14 institutions in 5 states) birth cohort study included children born at 23-27 weeks gestation in 2002-2004 who were evaluated for ASD and ID at age 10 years. Pregnancy information was obtained from medical records and by structured maternal interview. Cervical-vaginal ‘infection’ refers to maternal report of bacterial infection (n = 4), bacterial vaginosis (n = 30), yeast infection (n = 62), mixed infection (n = 4) or other/unspecified infection (n=43; e.g., chlamydia, trichomonas or herpes, etc.). We do not know the extent to which ‘infection’ per se was confirmed by microbial colonization. We use the terms ‘fetal growth restriction’ and ‘small for gestational age’ interchangeably in light of the ongoing challenge to discern pathologically from constitutionally small newborns. Severe fetal growth-restriction was defined as a birth weight Z-score for gestational age at delivery < - 2 (i.e., 2 standard deviations or more below the median birth weight in a referent sample that excluded pregnancies delivered for preeclampsia or fetal indications). Participants were classified into four groups based on whether or not they met rigorous diagnostic criteria for ASD and ID (ASD+/ID−, ASD+/ID+, ASD−/ID+ and ASD−/ID−). Temporally-ordered multinomial logistic regression models were used to examine the information conveyed by perinatal factors about increased risk for ASD and/or ID (ASD+/ID−, ASD+/ID+ and ASD−/ID+). RESULTS 889 of 966 (92%) children recruited were assessed at age 10 years, of whom 857 (96%) were assessed for ASD; of these, 840 (98%) children were assessed for ID. ASD+/ID− was diagnosed in 3.2% (27/840), ASD+/ID+ in 3.8% (32/840), and ASD−/ID+ in 8.5% (71/840). Maternal report of presumed cervical-vaginal ‘infection’ during pregnancy was associated with increased risk of ASD+/ID+ (odd ratio [OR], 2.7; 95% CI, 1.2-6.4). The lowest gestational age category (23-24 weeks) was associated with increased risk of ASD+/ID+ (OR, 2.9; 95% CI, 1.3-6.6) and ASD+/ID− (OR, 4.4; 95% CI, 1.7-11). Severe fetal growth restriction was strongly associated with increased risk for ASD+/ID− (OR, 9.9; 95% CI, 3.3-30), whereas peripartum maternal fever was uniquely associated with increased risk of ASD−/ID+ (OR, 2.9; 95% CI, 1.2-6.7). CONCLUSION Our study confirms that low gestational age is associated with increased risk for ASD irrespective of intellectual ability, whereas severe fetal growth restriction is strongly associated with ASD without ID. Maternal report of cervical-vaginal infection is associated with increased risk of ASD with ID, and per...

Objective To evaluate the difference in 10-year neurocognitive outcomes among extremely low gestational age newborns without bacteremia or with suspected or confirmed late-onset bacteremia. Study design Neurocognitive function was evaluated at 10 years of age in 889 children born at <28 weeks of gestation and followed from birth. Definite (culture positive) late-onset bacteremia during postnatal weeks 2–4 was identified in 223 children and 129 had suspected bacteremia. Results Infants with the lowest gestational age and birth weight Z-score had the highest prevalence of definite and suspected late-onset bacteremia. When compared with peers with no or suspected bacteremia, infants with definite bacteremia performed worse on tests of general cognitive ability, language, academic achievement, and executive function, even when adjusting for potential confounders. Adjustment for low IQ attenuated associations between bacteremia and all dysfunctions at 10 years. Children who had suspected bacteremia did not differ appreciably from children who did not have any evidence of bacteremia. The motor domain was unaffected. Conclusions Extremely low gestational age newborns who had definite late bacteremia during postnatal weeks 2–4 are at heightened risk of neurocognitive limitations at 10 years of age.

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Objectives-To examine elevated neonatal inflammatory and neurotrophic proteins from children born extremely preterm in relation to later childhood brain Magnetic Resonance Imaging volumes and cognition.Study design-We measured circulating inflammation-related proteins and neurotrophic proteins on postnatal days 1, 7, and 14 in 166 children at 10 years of age (73 males; 93 females). Top quartile levels on ≥2 days for ≥3 inflammation-related proteins and for ≥4 neurotrophic

Three quarters of children had normal intellect at age ten years; nearly 70% were free of neurodevelopmental impairment. Forty percent of children with impairments had multiple diagnoses.

The multiplicity of risk factors identified can be subsumed as components of four broad themes: low socioeconomic state, immaturity or vulnerability, inflammation, and epigenetic phenomena.