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BACKGROUND: To unravel the evolutionary history of a complex group, a comprehensive reconstruction of its phylogenetic relationships is crucial. This requires meticulous taxon sampling and careful consideration of multiple characters to ensure a complete and accurate reconstruction. The phylogenetic position of the Orestias genus has been estimated partly on unavailable or incomplete information. As a consequence, it was assigned to the family Cyprindontidae, relating this Andean fish to other geographically distant genera distributed in the Mediterranean, Middle East and North and Central America. In this study, using complete genome sequencing, we aim to clarify the phylogenetic position of Orestias within the Cyprinodontiformes order. RESULTS: We sequenced the genome of three Orestias species from the Andean Altiplano. Our analysis revealed that the small genome size in this genus (~ 0.7 Gb) was caused by a contraction in transposable element (TE) content, particularly in DNA elements and short interspersed nuclear elements (SINEs). Using predicted gene sequences, we generated a phylogenetic tree of Cyprinodontiformes using 902 orthologs extracted from all 32 available genomes as well as three outgroup species. We complemented this analysis with a phylogenetic reconstruction and time calibration considering 12 molecular markers (eight nuclear and four mitochondrial genes) and a stratified taxon sampling to consider 198 species of nearly all families and genera of this order. Overall, our results show that phylogenetic closeness is directly related to geographical distance. Importantly, we found that Orestias is not part of the Cyprinodontidae family, and that it is more closely related to the South American fish fauna, being the Fluviphylacidae the closest sister group. CONCLUSIONS: The evolutionary history of the Orestias genus is linked to the South American ichthyofauna and it should no longer be considered a member of the Cyprinodontidae family. Instead, we submit that Orestias belongs to the Orestiidae family, as suggested by Freyhof et al. (2017), and that it is the sister group of the Fluviphylacidae family, distributed in the Amazonian and Orinoco basins. These two groups likely diverged during the Late Eocene concomitant with hydrogeological changes in the South American landscape.

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BACKGROUND: The diversity and population genetic structure of many species have been shaped by historical and contemporary climatic changes. For the species of the South American Altiplano, the historical climatic changes are mainly related to the wet events of great magnitude and regional influence that occurred during the Pleistocene climatic oscillations (PCOs). In contrast, contemporary climate changes are associated with events of lesser magnitude and local influence related to intensifications of the South American Summer Monsoon (SASM). Although multiple studies have analyzed the effect of PCOs on the genetic patterns of highland aquatic species, little is known about the impact of contemporary climate changes in recent evolutionary history. Therefore, in this study, we investigated the change in population structure and connectivity using nuclear and mitochondrial markers throughout the distribution range of Heleobia ascotanensis, a freshwater Cochliopidae endemic to the Ascotán Saltpan. In addition, using geometric morphometric analyses, we evaluated the concomitance of genetic divergence and morphological differentiation. RESULTS: The mitochondrial sequence analysis results revealed the presence of highly divergent co-distributed and geographically nested haplotypes. This pattern reflects an extension in the distribution of groups that previously would have differentiated allopatrically. These changes in distribution would have covered the entire saltpan and would be associated with the large-scale wet events of the PCOs. On the other hand, the microsatellite results defined five spatially isolated populations, separated primarily by geographic barriers. Contemporary gene flow analyses suggest that post-PCO, climatic events that would have connected all populations did not occur. The morphometric analyses results indicate that there is significant morphological differentiation in the populations that are more isolated and that present the greatest genetic divergence. CONCLUSIONS: The contemporary population structure and morphological variation of H. ascotanensis mainly reflect the post-PCO climatic influence. Although both markers exhibit high genetic structuring, the microsatellite and morphology results show the preponderant influence of fragmentation in recent evolutionary history. The contemporary genetic pattern shows that in species that have limited dispersal capabilities, genetic discontinuities can appear rapidly, erasing signs of historical connectivity.

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Desert aquatic species tend to show isolated and disconnected populations due to the fragmented nature of their environment; however, the morphology of the hydrographic basins, added to humid climatic conditions, can allow dispersion between populations in a desert environment. The aim of this study was to examine the influence of drainage morphology on the phylogeographic structure and gene flow (using a fragment of the mitochondrial control region and seven microsatellite markers) of an endemic taxon of the Andean Precordillera in the Atacama Desert, the aquatic frog species Telmatobius pefauri. We detected three genetic clusters, one cluster present in the Lluta basin and two clusters in the Azapa basin. The results suggest that the genetic structure of T. pefauri is influenced by the morphology of the drainage network formed by the Lluta and Azapa basins: localities present in the same drainage, Tignamar River, were less differentiated and showed higher gene flow levels among them than to their conspecifics belonging to the other drainage in the same basin, Seco River, and those belonging to the other basin, Lluta basin. Gene flow patterns and genetic structure to populations Atacama Andean aquatic taxa would be influenced by basin morphology, with dispersion being stimulated in dendritic hydrological systems, and eventually by humid climatic (regional) events.

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Orestias ascotanensis (Cyprinodontidae) is a teleost pupfish endemic to springs feeding into the Ascotan saltpan in the Chilean Altiplano (3,700 m.a.s.l.) and represents an opportunity to study adaptations to high-altitude aquatic environments. We have de novo assembled the genome of O. ascotanensis at high coverage. Comparative analysis of the O. ascotanensis genome showed an overall process of contraction, including loss of genes related to G-protein signaling, chemotaxis and signal transduction, while there was expansion of gene families associated with microtubule-based movement and protein ubiquitination. We identified 818 genes under positive selection, many of which are involved in DNA repair. Additionally, we identified novel and conserved microRNAs expressed in O. ascotanensis and its closely-related species, Orestias gloriae. Our analysis suggests that positive selection and expansion of genes that preserve genome stability are a potential adaptive mechanism to cope with the increased solar UV radiation to which high-altitude animals are exposed to.

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Morphometrics has been used on Triatomines, a well-known phenotypically variable insect, to understand the process of morphological plasticity and infer the changes of this phenomenon. The following research was carried out in two regions of the inter-Andean valleys and two Chaco regions of Chuquisaca-Bolivia. Triatoma infestans adults were collected from the peridomestic (pens and chicken coops) along a geographic gradient in order to evaluate the morphological differentiation between groups and their pattern of sexual shape dimorphism. Geometric morphometric methods were applied on the wings and heads of T. infestans. The main findings include that we proved sexual dimorphism in heads and wings, determined the impact of environmental factors on size and shape and validated the impact of nutrition on head shape variation. These results show that geometric morphometric procedures can be used to provide key insight into the biological adaptation of T. infestans on different biotic (nutrition) and abiotic (environment) conditions, which could serve in understanding and evaluating infestation processes and further vector control programs.

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BACKGROUND: The causes of geographic variation of body size in ectotherms have generally been attributed to environmental variables. Research in amphibians has favored mechanisms that involve water availability as an explanation for the geographic variation of body size. However, there are few studies at intraspecific level on amphibians that inhabit desert or semi-desert environments, where hydric restrictions are stronger. Here, we describe and inquire as to the causes of the geographic variation of body size in the semi-desert toad Rhinella atacamensis, a terrestrial anuran that is distributed over 750 km along a latitudinal aridity gradient from the southern extreme of the Atacama Desert to the Mediterranean region of central Chile. We measured the snout-vent length of 315 adults from 11 representative localities of the entire distribution of the species. Then, using an information-theoretic approach, we evaluate whether the data support eight ecogeographic hypotheses proposed in literature. RESULTS: Rhinella atacamensis exhibits a gradual pattern of decrease in adult body size towards the north of its distribution, where the climate is more arid, which conforms to a Bergmann's cline. The best model showed that the data support the mean annual precipitation as predictor of body size, favoring the converse water availability hypothesis. CONCLUSIONS: Most studies in amphibians show that adult size increases in arid environments, but we found a converse pattern to expected according to the hydric constraints imposed by this type of environment. The evidence in R. atacamensis favors the converse water availability hypothesis, whose mechanism proposes that the foraging activity determined by the precipitation gradient has produced the clinal pattern of body size variation. The variation of this trait could also be affected by the decreasing productivity that exists towards the north of the species distribution. In addition, we found evidence that both pattern and mechanism are independent of sex. Lastly, we suggest that behavioral traits, such as nocturnal habits, might also play an important role determining this differential response to aridity. Therefore, the support for the converse water availability hypothesis found in this study shows that amphibians can respond in different ways to water restrictions imposed by arid environments.

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The morphological variations of four populations of geographically isolated Triatoma infestans located in the area of inter-Andean valleys and Chaco of Chuquisaca, Bolivia, were evaluated. Fifty-three females and sixty-one males were collected in the peri-domicile and analyzed with geometric morphometrics tools to study the patterns of the head and wing shape variation. The principal component analysis and canonical variate analysis revealed morphological variations between the populations studied, which were then confirmed by the permutation test of the differences between populations using Mahalanobis and Procustes distances. The multivariate regression analysis shows that the centroid size influences the shape of the heads and wings. T. infestans of the inter-Andean valleys are longer in the head and wings compared to the population of the Chaco. We propose that the geometric shape variation may be explained by geographical changes in climatic conditions, peri-domiciliar habitats, food source quality, and the use of insecticides.

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Sweet cherry is a valuable non-climacteric fruit with elevated phytonutrients, whose fruit quality attributes are prone to rapid deterioration after harvest, especially peel damage and water loss of stem. Here the metabolic and transcriptional response of exogenous melatonin was assessed in two commercial cultivars of sweet cherry (Santina and Royal Rainier) during cold storage. Gene expression profiling revealed that cuticle composition and water movement may underlie the effect of melatonin in delaying weight loss. An effect of melatonin on total soluble solids and lower respiration rate was observed in both cultivars. Melatonin induces overexpression of genes related to anthocyanin biosynthesis, which correlates with increased anthocyanin levels and changes in skin color (Chroma). Our results indicate that along with modulating antioxidant metabolism, melatonin improves fruit quality traits by triggering a range of metabolic and gene expression changes, which ultimately contribute to extend sweet cherry postharvest storability.

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The morphological variations of four populations of geographically isolated Triatoma infestans located in the area of inter-Andean valleys and Chaco of Chuquisaca, Bolivia, were evaluated. Fifty-three females and sixty-one males were collected in the peri-domicile and analyzed with geometric morphometrics tools to study the patterns of the head and wing shape variation. The principal component analysis and canonical variate analysis revealed morphological variations between the populations studied, which were then confirmed by the permutation test of the differences between populations using Mahalanobis and Procustes distances. The multivariate regression analysis shows that the centroid size influences the shape of the heads and wings. T. infestans of the inter-Andean valleys are longer in the head and wings compared to the population of the Chaco. We propose that the geometric shape variation may be explained by geographical changes in climatic conditions, peri-domiciliar habitats, food source quality, and the use of insecticides.

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BACKGROUND: The origin of birds is marked by a significant decrease in body size along with an increase in relative forelimb size. However, before the evolution of flight, both traits may have already been related: It has been proposed that an evolutionary trend of negative forelimb allometry existed in non-avian Theropoda, such that larger species often have relatively shorter forelimbs. Nevertheless, several exceptions exist, calling for rigorous phylogenetic statistical testing. RESULTS: Here, we re-assessed allometric patterns in the evolution of non-avian theropods, for the first time taking into account the non-independence among related species due to shared evolutionary history.We confirmed a main evolutionary trend of negative forelimb allometry for non-avian Theropoda, but also found support that some specific subclades (Coelophysoidea, Ornithomimosauria, and Oviraptorosauria) exhibit allometric trends that are closer to isometry, losing the ancestral negative forelimb allometry present in Theropoda as a whole. CONCLUSIONS: Explanations for negative forelimb allometry in the evolution of non-avian theropods have not been discussed, yet evolutionary allometric trends often reflect ontogenetic allometries, which suggests negative allometry of the forelimb in the ontogeny of most non-avian theropods. In modern birds, allometric growth of the limbs is related to locomotor and behavioral changes along ontogeny. After reviewing the evidence for such changes during the ontogeny of non-avian dinosaurs, we propose that proportionally longer arms of juveniles became adult traits in the small-sized and paedomorphic Aves.

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Phosphorylation of the human α1B-adrenergic receptor (fused with the green fluorescent protein) was studied employing the inducible Flp-ln HEK293 T-Rex system for expression. Serine/alanine substitutions were performed in five sites corresponding to those previously identified as phosphorylation targets in the hamster ortholog. Desensitization was decreased in these mutants but receptor phosphorylation was still clearly detected. The protein phosphorylation of the wild-type receptor (fused to the green fluorescent protein) was studied, using mass spectrometry, under baseline and stimulated conditions (noradrenaline or phorbol myristate acetate). Basal phosphorylation was detected at sites located at the intracellular loop 3 and carboxyl terminus, and the number of sites detected increased under agonist activation and stimulation of protein kinase C. The phosphorylation patterns differed under the distinct conditions. Three of the phosphorylation sites detected in this work corresponded to those observed in the hamster receptor. The phosphorylation sites detected included the following: a) at the intracellular loop 3: serines 246, 248, 257, 267, and 277; and threonines 252, 264, and 268, and b) at the carboxyl terminus: serines 396, 400, 402, 406, 423, 425, 427, 455, and 470, and threonines 387, 392, 420, and 475. Our data indicate that complex phosphorylation patterns exist and suggest the possibility that such differences could be relevant in receptor function and subcellular localization.

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Species delimitation in minute freshwater snails is often difficult to perform using solely shell morphology. The problem intensifies when invasive species spread within the distribution range of morphologically similar native species. In Chile, the Truncatelloidean snails are represented by the native genera Heleobia and Potamolithus plus the invasive mudsnail Potamopyrgus antipodarum, which can easily be confused. Using an integrative approach, we performed molecular phylogenetic analysis and studied reproductive and morphological features to identify superficially similar forms inhabiting the central area of the country. Truncatelloidean snails were identified in 40 of 51 localities sampled, 10 containing Potamopyrgus antipodarum, 23 Heleobia and 7 Potamolithus. Based on these results and previously published data, the known distribution of the mudsnail in Chile encompasses 6 hydrological basins, including 18 freshwater ecosystems. The finding of the mudsnails in several type localities of native species/subspecies of "Heleobia" that were not find in situ suggests species replacement or significant extinction of native fauna, a hypothesis supported by the restudy of type material that shows that endemic forms belong to the genus Potamolithus. This study shows the usefulness of integrative taxonomy not only resolving complex taxa with cryptic morphology but also measuring the extent of an ongoing invasion.

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The human α1D-adrenergic receptor is a seven transmembrane-domain protein that mediates many of the physiological actions of adrenaline and noradrenaline and participates in the development of hypertension and benign prostatic hyperplasia. We recently reported that different phosphorylation patterns control α1D-adrenergic receptor desensitization. However, to our knowledge, there is no data regarding the role(s) of this receptor's specific phosphorylation residues in its subcellular localization and signaling. In order to address this issue, we mutated the identified phosphorylated residues located on the third intracellular loop and carboxyl tail. In this way, we experimentally confirmed α1D-AR phosphorylation sites and identified, in the carboxyl tail, two groups of residues in close proximity to each other, as well as two individual residues in the proximal (T442) and distal (S543) regions. Our results indicate that phosphorylation of the distal cluster (T507, S515, S516 and S518) favors α1D-AR localization at the plasma membrane, i. e., substitution of these residues for non-phosphorylatable amino acids results in the intracellular localization of the receptors, whereas phospho-mimetic substitution allows plasma membrane localization. Moreover, we found that T442 phosphorylation is necessary for agonist- and phorbol ester-induced receptor colocalization with ß-arrestins. Additionally, we observed that substitution of intracellular loop 3 phosphorylation sites for non-phosphorylatable amino acids resulted in sustained ERK1/2 activation; additional mutations in the phosphorylated residues in the carboxyl tail did not alter this pattern. In contrast, mobilization of intracellular calcium and receptor internalization appear to be controlled by the phosphorylation of both third-intracellular-loop and carboxyl terminus-domain residues. In summary, our data indicate that a) both the phosphorylation sites present in the third intracellular loop and in the carboxyl terminus participate in triggering calcium signaling and in turning-off α1D-AR-induced ERK activation; b) phosphorylation of the distal cluster appears to play a role in receptor's plasma membrane localization; and c) T442 appears to play a critical role in receptor phosphorylation and receptor-ß-arrestin colocalization.

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In the present study, we focus on the phylogeographic pattern, demographic history and morphological differentiation of Heleobia ascotanensis, a freshwater gastropod restricted to the Ascotán saltpan in the Chilean Altiplano. The current distribution of the species is limited to twelve isolated or partially isolated springs that were affected by transitions between humid and arid periods during last glaciations. The genetic analysis of 322 specimens showed that H. ascotanensis is subdivided into three genetically divergent populations, with low and moderate degrees of historical gene flow among them and incipient morphological differentiation as a consequence of genetic and geographical isolation. Molecular analyses revealed different demographic histories among populations which seem to respond independently to climatic events, probably due to an environmental imposition and idiosyncratic strategies developed to cope with water availability. The results of this study and co-distributed taxa support the hypothesis that contemporary and historical events have influenced microevolutionary differentiation of these snails, although there is a need to complement further information to predict genetic or morphological divergence at microgeographic scale.

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The genus Telmatobius Wiegmann, 1834 is composed of a wide variety of species and is one of the most diverse native components of the high-altitude Andean environments. The species of the genus present in Chile are considered as endangered, critically endangered or data deficient. We isolated and evaluated 44 microsatellites in 80 individuals of 8 species of Telmatobius present in Chile, obtaining 22 polymorphic microsatellite loci for Telmatobius chusmisensis. The cross-amplification test was successful in all other species tested. For the first time, microsatellite markers are described for Telmatobius. The description of these primers will be useful for further genetic studies for T. chusmisensis and other species of the same genus; allowing further analyses of population structuring, dispersal patterns, recent demographic history and population effective size. This information is also significant to undertake conservation actions for the species of the genus Telmatobius, since most species have conservation issues.

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The origin of cryptic species has traditionally been associated with events of recent speciation, genetic constraints, selection of an adaptive character, sexual selection and/or convergent evolution. Species of the genus Callyntra inhabit coastal terraces, mountain slopes, and peaks; their elytral designs are associated with each of these habitats. However, cryptic species have been described within each of these habitats; the taxonomy of this group has been problematic, thus establishing the phylogenetic relationships in this group is fundamental to clarify the systematics and evolutionary patterns of Callyntra. We reconstructed the phylogeny of this group using two mitochondrial genes (COI, 16S) and one nuclear gene (Mp20). We also performed species delimitation using PTP based methods (PTP, mlPTP, bPTP) and GMYC, and evaluated the evolution of the elytral design related to habitat preference. The results showed a tree with five clades, that together with the different methods of species delimitation recovered the described species and suggested at least five new species. The elytral design and habitat preference showed phylogenetic signals. We propose a new classification based on monophyletic groups recovered by phylogenetic analyses. We also suggest that parallel evolution in different habitats and later stasis in the elytral design would be the cause of the origin of cryptic species in this group from central Chile.

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Human α1D-adrenoceptors (α1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α1D-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/ß, phosphorylate α1D-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α1-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.

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Telmatobius halli was the first endemic Telmatobius species described in Chile, reported by Noble in 1938 near the locality of Ollagüe, in the high Andean zone of the Antofagasta region. To this date, there are no specimens assignable to this species other than the type series; although many expeditions have tried to search for T. halli, they have been unsuccessful, but they have found and described new species around this area. In order to clarify the origin of the enigmatic T. halli, we reviewed the itinerary of the expedition carried out by F. G. Hall in the Chilean Altiplano, to place a putative type locality. We contrast the morphology of the holotype, with that of recently collected specimens from the new putative type locality, to confirm the population's identity; and finally, we perform phylogenetic analyses in order to clarify the systematic position of this taxon. The historical review of the expedition that collected these frogs shows that it is likely that Telmatobius halli had been collected near Collahuasi, about 50 km northwest of Ollagüe, site that we have assigned as a putative type locality for T. halli. The morphological analyses support this hypothesis, while phylogenetic results show that the specimens assigned to this species form a monophyletic group, and is a sister clade of T. chusmisensis. Thus, we propose that the type locality of T. halli be changed from "around Ollagüe" to the area of the Copaquire ravine, so its distribution would be restricted to this system and Choja-Chijlla ravine, both in the high Andean zone of the Región de Tarapacá, Chile.

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Orestias Valenciennes, 1839 is a genus of freshwater fish endemic to the South American Altiplano. Cytogenetic studies of these species have focused on conventional karyotyping. The aim of this study was to use classical and molecular cytogenetic methods to identify the constitutive heterochromatin distribution and chromosome organization of four classes of repetitive DNA sequences (histone H3 DNA, U2 snRNA, 18S rDNA and 5S rDNA) in the chromosomes of O. ascotanensis Parenti, 1984, an endemic species restricted to the Salar de Ascotán in the Chilean Altiplano. All individuals analyzed had a diploid number of 48 chromosomes. C-banding identified constitutive heterochromatin mainly in the pericentromeric region of most chromosomes, especially a GC-rich heterochromatic block of the short arm of pair 3. FISH assay with an 18S probe confirmed the location of the NOR in pair 3 and revealed that the minor rDNA cluster occurs interstitially on the long arm of pair 2. Dual FISH identified a single block of U2 snDNA sequences in the pericentromeric regions of a subtelocentric chromosome pair, while histone H3 sites were observed as small signals scattered in throughout the all chromosomes. This work represents the first effort to document the physical organization of the repetitive fraction of the Orestias genome. These data will improve our understanding of the chromosomal evolution of a genus facing serious conservation problems.

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In LNCaP cells that stably express α1A-adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α1A-Adrenergic receptor interaction with ß-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α1A-adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α1A-AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined.

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