Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.

The intrahelical salt bridge between E/D(3.49) and R(3.50) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E(3.49/6.30) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

Nonequilibrium activation of a G-protein-coupled receptor.

G-protein-coupled receptor activation is generally analyzed under equilibrium conditions. However, real-life receptor functions are often dependent on very short, transient stimuli that may not allow the achievement of a steady state. This is particularly true for synaptic receptors such as the α(2A)-adrenergic receptor (α(2A)-AR). Therefore, we developed a fluorescence resonance energy transfer-based technology to study nonequilibrium α(2A)-AR function in living cells. To examine the effects of increasing concentrations of the endogenous agonist norepinephrine on the speed and extent of α(2A)-AR activation with very high temporal resolution, we took advantage of a fluorophore-containing α(2A)-AR sensor. The results indicated that the efficacy of norepinephrine in eliciting receptor activation increased in a time-dependent way, reaching the maximum with a half-life of ~60 ms. The EC(50) values under nonequilibrium conditions start at ~26 µM (at 40 ms) and show a 10-fold decrease until the steady state is achieved. To analyze the ability of norepinephrine to trigger a downstream intracellular response after α(2A)-AR stimulation, we monitored the kinetics and amplitude of G(i) activation in real time by using a fluorophore-containing G(i) sensor. The results show that both the efficacy and the potency of norepinephrine in inducing G(i) activation achieve a steady state more slowly, compared with receptor activation, and that the initial EC(50) value of ~100 nM decreases in an exponential way, reaching the minimal value of ~10 nM at equilibrium. Therefore, both the efficacy and the potency of norepinephrine increase ~10-fold over a few seconds of agonist stimulation, which illustrates that receptor and G-protein signaling and signal amplification are highly time-dependent phenomena.

Light on the structure of thromboxane A2receptor heterodimers.

The structure-based design of a mutant form of the thromboxane A(2) prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the hetero-dimeric complexes between the TPα and ß isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPα-TPß hetero-dimerization serves to regulate TPα function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution.

Sensing G protein-coupled receptor activation.

G protein-coupled receptors (GPCRs) are the key elements of a highly regulated transduction machinery that generates different signaling outcomes to hormones and neurotransmitters. Until recently, it was assumed that diverse ligands of a given GPCR differ only in their ability to alter the balance between the OFF and the ON state of the receptor. However, it has now become evident that their activation mechanisms are more complex and that receptors presumably display distinguishable active conformational states, which are induced by different agonists and correlate to specific signaling outputs. The use of different labeling strategies to insert fluorescent labels into purified, reconstituted receptors, or into receptors in intact cells, has made it possible to sense receptor activation via changes in their fluorescence. Here, we summarize recent progress in the analysis of agonist-dependent activation mechanisms of GPCRs acquired using modern spectroscopic and crystallographic techniques.

Differential signaling of the endogenous agonists at the beta2-adrenergic receptor.

The concept of "functional selectivity" or "biased signaling" suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the ß(2)-adrenergic receptor (ß(2)AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged ß(2)AR FRET sensor. In this ß(2)AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard "full" agonist isoproterenol. Furthermore, norepinephrine-induced changes in the ß(2)AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial ß(2)AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G(s) and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering ß-arrestin2 recruitment to the cell surface and its interaction with ß(2)AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the ß(2)AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G(s) and less efficient in signaling to ß-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the ß(2)AR and link it to distinct conformational changes in the receptor.

Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants.

In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129((3.49)) or E240((6.30)) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G(q) and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM.

Práticas de amamentação de puérparas na consulta de enfermagem neonatal em Unidade Básica de Saúde / Breastfeeding practices among postpartum women during nursing consultations in a primary health care center / Prácticas de lactancia materna entre puérperas en la consulta de enfermería neonatal de una unidad básica de salud

Estudo descritivo-exploratório, cujos objetivos foram averiguar a prática da amamentação de puérperas que levaram o filho recém-nascido na unidade básica de saúde e avaliar os passos para o sucesso da amamentação durante a consulta de enfermagem. A amostra reuniu 13 puérperas que levaram seu filho para a consulta de enfermagem em uma unidade básica de saúde no município do Rio de Janeiro. A coleta de dados foi realizada mediante um formulário. Os resultados evidenciam que 84,6% (11) referiram que os bebês mamam em ambos os seios, 76,9% (10) oferecem mamadas com frequência, 38,4% (5) responderam que a duração das mamadas foi de aproximadamente 20 minutos. Recomenda-se manter uma rede de apoio à puérpera, ao recém-nascido e à sua família para o sucesso do aleitamento materno.

This is a descriptive and exploratory study that aims to assess the breastfeeding practices among postpartum women in a primary health care center, as well as to evaluate the steps for a successful breastfeeding during a nursing consultation. The sample was composed by 13 women who took their children to a nursing consultation in a primary health care center in the city of Rio de Janeiro. A specific form was used to collect data. Results show that 84.6% (n=11) of the patients used both breasts to feed their babies; 76.9% (n=10) offered unrestricted breastfeeding, and 38.4% (n=5) referred that breastfeeding time was approximately 20 minutes. It is recommended to maintain a support network for postpartum women, newborns and their families to provide a successful breastfeeding.

Estudio exploratorio descriptivo realizado con el objetivo de investigar la práctica de lactancia materna entre puérperas que llevaron a su hijo recién nacido a una unidad básica de salud y evaluar los pasos para amamantar correctamente durante la consulta de enfermería. La muestra reunió a 13 mujeres que llevaron a su hijo para la consulta de enfermería a una unidad básica de salud del municipio de Rio de Janeiro, con recogida de datos en un formulario. Los resultados indicaron que 84,6% (11) informó que los niños se alimentaban de los dos pechos, 76,9% (10) ofrecían el pecho con frecuencia, el 38,4% (5) respondió que la duración de la lactancia era de cerca de 20 minutos. Se recomienda mantener una red de apoyo a la puérpera, al recién nacido y a su familia para que la lactancia materna se lleve a cabo con éxito.