Gregory N. Prado

Role of the C Terminus of the Interleukin 8 Receptor in Signal Transduction and Internalization

Interleukin 8 (IL-8) is a potent neutrophil chemoattractant and activator. Two IL-8 receptor subtypes, A and B, are expressed in neutrophils. In this work, we analyzed the role of the C terminus domain of the IL-8 receptor on the signal transduction and receptor internalization mechanisms. The IL-8 receptor A was tagged with an epitope corresponding to the monoclonal antibody 1D4 to monitor the localization of the IL-8 receptor. We demonstrated IL-8-dependent receptor internalization by monitoring the density of surface 125I-labeled IL-8 binding sites and by immunofluorescence microscopy. Truncation of the last 27 amino acids of the IL-8 receptor A severely impaired the IL-8-induced internalization of the receptor. Of importance was the observation that binding of IL-8 to receptors A and B triggered a dramatically faster rate of internalization of receptor B than receptor A, suggesting that the heterologous C termini among receptor subtypes modulate the rate of internalization of IL-8 receptors. However, substitution of the C terminus of the receptor subtype A for the C terminus of receptor B reduced the internalization rate of receptor A. Furthermore, we found that the rate of internalization of IL-8 receptor B triggered by IL-8 was faster than the one induced by the IL-8-related peptide, melanoma growth stimulatory activity. Studies with human neutrophils pretreated with 100 nM IL-8 for 5 min revealed a positive and a negative calcium response mediated by receptors A and B, respectively. In contrast, neutrophils pretreated with melanoma growth stimulatory activity showed positive calcium responses to both receptors A and B. These data suggest that the neutrophil responses mediated by IL-8 are modulated by the rate of internalization of receptors.

Aldosterone's Rapid, Nongenomic Effects Are Mediated by Striatin: A Modulator of Aldosterone's Effect on Estrogen Action

The cellular responses to steroids are mediated by 2 general mechanisms: genomic and rapid/nongenomic effects. Identification of the mechanisms underlying aldosterone (ALDO)s rapid vs their genomic actions is difficult to study, and these mechanisms are not clearly understood. Recent data suggest that striatin is a mediator of nongenomic effects of estrogen. We explored the hypothesis that striatin is an intermediary of the rapid/nongenomic effects of ALDO and that striatin serves as a novel link between the actions of the mineralocorticoid and estrogen receptors. In human and mouse endothelial cells, ALDO promoted an increase in phosphorylated extracellular signal-regulated protein kinases 1/2 (pERK) that peaked at 15 minutes. In addition, we found that striatin is a critical intermediary in this process, because reducing striatin levels with small interfering RNA (siRNA) technology prevented the rise in pERK levels. In contrast, reducing striatin did not significantly affect 2 well-characterized genomic responses to ALDO. Down-regulation of striatin with siRNA produced similar effects on estrogens actions, reducing nongenomic, but not some genomic, actions. ALDO, but not estrogen, increased striatin levels. When endothelial cells were pretreated with ALDO, the rapid/nongenomic response to estrogen on phosphorylated endothelial nitric oxide synthase (peNOS) was enhanced and accelerated significantly. Importantly, pretreatment with estrogen did not enhance ALDOs nongenomic response on pERK. In conclusion, our results indicate that striatin is a novel mediator for both ALDOs and estrogens rapid and nongenomic mechanisms of action on pERK and phosphorylated eNOS, respectively, thereby suggesting a unique level of interactions between the mineralocorticoid receptor and the estrogen receptor in the cardiovascular system.

Endothelin-1 receptor antagonists regulate cell surface-associated protein disulfide isomerase in sickle cell disease

Increased endothelin‐1 (ET‐1) levels, disordered thiol protein status, and erythrocyte hydration status play important roles in sickle cell disease (SCD) through unresolved mechanisms. Protein disulfide isomerase (PDI) is an oxidoreductase that mediates thiol/disulfide interchange reactions. We provide evidence that PDI is present in human and mouse erythrocyte membranes and that selective blockade with monoclonal antibodies against PDI leads to reduced Gardos channel activity (1.6±0.03 to 0.56±0.02 mmol·1013 cell–1 ·min–1, P< 0.001) and density of sickle erythrocytes (D50: 1.115±0.001 to 1.104±0.001 g/ml, P=0.012) with an IC50 of 4 ng/ml. We observed that erythrocyte associated‐PDI activity was increased in the presence of ET‐1 (3.1 ± 0.2 to 5.6 ± 0.4%, P<0.0001) through a mechanism that includes casein kinase II. Consistent with these results, in vivo treatment of BERK sickle transgenic mice with ET‐1 receptor antagonists lowered circulating and erythrocyte associated‐PDI activity (7.1±0.3 to 5.2±0.2%, P<0.0001) while improving hematological parameters and Gardos channel activity. Thus, our results suggest that PDI is a novel target in SCD that regulates erythrocyte volume and oxidative stress and may contribute to cellular adhesion and endothelial activation leading to vasoocclusion as observed in SCD.—Prado, G. N., Romero, J. R., Rivera, A. Endothelin‐1 receptor antagonists regulate cell surface‐associated protein disulfide isomerase in sickle cell disease. FASEB J. 27, 4619–4629 (2013). www.fasebj.org

Adenovirus mediated expression “in vivo” of the chemokine receptor CXCR1

A major hurdle in the structural analysis of membrane proteins is the expression of a functional and homogeneous form of the protein. Except for rhodopsin, most G protein-coupled receptors (GPCRs) are endogenously expressed at very low levels. Heterologous expression of GPCRs in bacteria, yeast, insect cells or mammalian cell lines often yields proteins with large amounts of misfolded proteins and heterogeneous posttranslational modifications. Here, we report a novel mammalian “in vivo” system for the expression of the chemokine receptor CXCR1. This receptor was expressed in liver of mice infected with adenovirus encoding CXCR1. Liver plasma membranes from infected mice displayed high-levels of 125I-labeled human interleukin-8 (IL-8) binding. The pharmacological profile of the recombinant CXCR1 expressed “in vivo” was similar to those expressed in neutrophils. We found that the incorporation of the detergent solubilized CXCR1 into phospholipid vesicles in the presence of Gi/Go proteins is required for the reconstitution of 125I-IL-8 binding. On the basis of the presence of the several endogenous His residues and glycosylation moieties in CXCR1 we fractionated the detergent-solubilized plasma membranes by employing Ni- and Concanavalin A-based chromatography. Fractions enriched with CXCR1 were monitored by 125I-IL-8-bound to the receptor and Western blots with anti-CXCR1 antibodies. This robust expression system could be readily applied for the expression of GPCRs and other eukaryotic membrane proteins.