In Vitro Selection of State-Specific Peptide Modulators of G Protein Signaling Using mRNA Display†

Biochemistry ◽  
2004 ◽  
Vol 43 (28) ◽  
pp. 9265-9275 ◽  
Author(s):  
William W. Ja ◽  
Richard W. Roberts
Keyword(s):  
G Protein ◽  
In Vitro Selection ◽  
G Protein Signaling ◽  
Mrna Display ◽  
Protein Signaling ◽  
Specific Peptide ◽  
Selection Of

scholarly journals Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes

2021 ◽  
Vol 14 (673) ◽  
pp. eaax3053
Author(s):  
Mieke Metzemaekers ◽  
Anneleen Mortier ◽  
Alessandro Vacchini ◽  
Daiane Boff ◽  
Karen Yu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Chemotactic Activity ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Agonist Activity ◽  
N Terminus ◽  
G Protein Coupled

The inflammatory human chemokine CXCL5 interacts with the G protein–coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1–78), truncated CXCL5 [CXCL5(9–78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and β-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

181 THE POTENTIAL ROLE OF NON-GENOMIC PATHWAYS AND THEIR EFFECTS ON THE REGULATION OF CALBINDIN-D9k IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 189
Author(s):  
V. H. Dang ◽  
E.-B. Jeung
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Gh3 Cells ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Calbindin D9k ◽  
Protein Expressions ◽  
Genomic Effects

Calbindin-D9k (CaBP-9k), a cytosolic protein, is one of the members of the family of vitamin D-dependent calcium-binding proteins with high affinity for calcium. The previous in vitro studies indicated that this gene is controlled by 17β-estradiol (E2), a physiological estrogen, via both genomic (through its classical nuclear receptors) and non-genomic (through different cypoplasmic signals) mechanisms. In order to provide a better understanding in molecular events by which E2 exerts its actions in the regulation of CaBP-9k, we employed GH3 cells as an in vitro model to examine the possible non-genomic effects of E2 on the induction of CaBP-9k. GH3 cells were treated dose-dependently (10–5, 10–6, 10–7, 10–8, and 10–9 m) with E2-BSA, a membrane-impermeable E2 conjugated with BSA, for 24 h. To examine the time dependency, the cells were also exposed to a high concentration (10–6 m) of E2-BSA and harvested at various time points (5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h). Furthermore, in order to determine the potential involvement of non-genomic signaling pathways in E2-BSA-induced expression of CaBP-9k, several inhibitors also were employed, including ICI 182 780 for membrane estrogen receptor (ER) pathway, pertussis toxin (PTX) for G protein signaling, U0126 for ERK pathway, and wortmannin for Akt pathway. The non-genomic effects of E2-BSA on the induction of CaBP-9k mRNA and protein were determined by semi-quantitative RT-PCR and Western blotting, respectively. In a dose-dependent manner, administration with E2-BSA (10–6 m) induced the highest response of CaBP-9k at transcriptional (mRNA) level, whereas protein level of CaBP-9k peaked at E2-BSA concentration (10–7 m) at 24 h. In a time course, E2-BSA (10–6 m) exposure caused a significant increase in both CaBP-9k mRNA and protein expressions as early as 15 min and peaked at 24 h. Co-treatment with ICI 182 780 and PTX completely inhibited E2-BSA-induced CaBP-9k mRNA and protein expressions. Interestingly, although co-treatments with U0126 and/or wortmannin alone failed to attenuate the effects of E2-BSA, a combination of 2 inhibitors completely reversed E2-BSA-induced CaBP-9k expressions at both transcriptional (mRNA) and translational (protein) levels, suggesting their involvement in the regulation of CaBP-9k in GH3 cells. Taken together, these results demonstrate that various signaling pathways may be involved in E2-induced regulation of CaBP-9k in which membrane ER and G protein signaling pathways play a central role in non-genomic responses. Further in vitro experiments are required to elucidate additional details of the interaction of ERK and Akt pathways in the regulation of CaBP-9k in these cells, offering a new insight into the mode of E2 action in the pituitary gland of human and wildlife.

scholarly journals Regulator of G-Protein Signaling 5 Reduces HeyA8 Ovarian Cancer Cell Proliferation and Extends Survival in a Murine Tumor Model

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Molly K. Altman ◽  
Duy T. Nguyen ◽  
Santosh B. Patel ◽  
Jada M. Fambrough ◽  
Aaron M. Beedle ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
G Protein ◽  
Expression System ◽  
Tumor Biology ◽  
Pathological Examination ◽  
Ovarian Cancer Cells ◽  
G Protein Signaling ◽  
Protein Signaling

The regulator of G-protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiated by extracellular mediators and G-protein-coupled receptors. RGS5 has an interesting dual biological role. One functional RGS5 role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other functional role is to promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumor progression—whether it plays a pathogenic or protective role in ovarian tumor biology. We thus constructed an inducible gene expression system to achieve RGS5 expression in HeyA8-MDR ovarian cancer cells. Through this we observed that inducible RGS5 expression significantly reducesin vitroBrdU-positive HeyA8-MDR cells, although this did not correlate with a reduction in tumor volume observed using anin vivomouse model of ovarian cancer. Interestingly, mice bearing RGS5-expressing tumors demonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observed by pathological examination. Additionally, mice bearing RGS5-expressing tumors were less likely to have ulcerated tumors. Taken together, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the context of a multicomponent approach for modulating tumor progression.

scholarly journals Λειτουργικές αλληλεπιδράσεις πρωτεϊνών με υποδοχείς που συζεύγνυνται με G πρωτεΐνες

2012 ◽  
Author(s):  
Λεωνίδας Λεοντιάδης
Keyword(s):  
G Protein ◽  
G Protein Signaling ◽  
Protein Signaling

Οι οπιοειδείς υποδοχείς ανήκουν στους υποδοχείς που συζεύγνυνται με G πρωτεΐνες (GPCRs) και ρυθμίζουν την αναλγησία και την ανοχή-εξάρτηση σε ναρκωτικές ουσίες. Στη σηματοδότηση των GPCRs εμπλέκεται μια νέα οικογένεια πρωτεϊνών, οι RGS πρωτεΐνες (Regulators of G protein Signaling), που επιταχύνουν το ρυθμό υδρόλυσης του GΤΡ από τις Gα υπομονάδες, τερματίζοντας έτσι τη μετάδοση του ερεθίσματος. Σκοπός της διατριβής ήταν να μελετηθούν οι αλληλεπιδράσεις των μ- και δ-οπιοειδών υποδοχέων (μ-OR, δ-OR) με την RGS4 πρωτεΐνη και ο ρόλος της RGS4 στη σηματοδότησή τους. Πειράματα in vitro πρόσδεσης με τη χρήση GST-/MBP-χιμαιρικών πεπτιδίων απέδειξαν ότι η RGS4 προσδένεται στην τρίτη ενδοκυτταρική θηλιά και στο συντηρημένο τμήμα των καρβοξυτελικών άκρων των οπιοειδών υποδοχέων (μ-CT, δ-CT). Η RGS4 λειτουργεί επίσης ως ικρίωμα και προάγει την πρόσδεση της Gα υπομονάδας στο μ-CT, σχηματίζοντας ένα ετεροτριμερές σύμπλοκο RGS4–Gα–μ-CT. Πειράματα συν-ανοσοκατακρήμνισης έδειξαν ότι η RGS4 προάγει την αλληλεπίδραση των μ-OR, δ-OR με συγκεκριμένους υποτύπους Gα υπομονάδων, ανάλογα με την κατάσταση ενεργοποίησης των υποδοχέων. Λειτουργικά πειράματα έδειξαν ότι η RGS4 α) μειώνει την αναστολή της αδενυλικής κυκλάσης, αυξάνοντας τα ποσοστά του διαθέσιμου ενδοκυτταρικού cAMP, β) αναστέλλει τη φωσφορυλίωση των ERK κινασών, γ) συνεντοπίζεται με τους ενεργοποιημένους υποδοχείς. Για αυτές τις λειτουργικές επιδράσεις ευθύνεται το αμινοτελικό άκρο της RGS4. Τα αποτελέσματα της διατριβής δεικνύουν ότι η RGS4 πρωτεΐνη είναι ένα μόριο-κλειδί για την ρύθμιση και εξειδίκευση της σηματοδότησης των οπιοειδών υποδοχέων.

scholarly journals The RGS Protein Crg2 Regulates Pheromone and Cyclic AMP Signaling in Cryptococcus neoformans

2008 ◽  
Vol 7 (9) ◽  
pp. 1540-1548 ◽  
Author(s):  
Gui Shen ◽  
Yan-Li Wang ◽  
Amy Whittington ◽  
Lie Li ◽  
Ping Wang
Keyword(s):  
Cyclic Amp ◽  
Cryptococcus Neoformans ◽  
G Protein ◽  
Mitogen Activated Protein Kinase ◽  
Camp Signaling ◽  
Cell Swelling ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Protein Activity

ABSTRACT Crg1 and Crg2 are regulators of G-protein signaling homologs found in the human fungal pathogen Cryptococcus neoformans. Crg1 negatively regulates pheromone responses and mating through direct inhibition of Gα subunits Gpa2 and Gpa3. It has also been proposed that Crg2 has a role in mating, as genetic crosses involving Δcrg2 mutants resulted in formation of hyperfilaments. We found that mutation of Gpa2 and Gpa3 partially suppressed the hyperfilamentation, mutation of Gpa3 alleviated Δcrg2-specfic cell swelling, and mutation of the mitogen-activated protein kinase Cpk1 blocked both processes. These findings indicate that Gpa2 and Gpa3 function downstream of Crg2 and that Gpa3 is also epistatic to Crg2 in a Cpk1-dependent morphogenesis process linked to mating. Significantly, we found that Δcrg2 mutants formed enlarged capsules that mimic cells expressing a constitutively active GPA1(Q284L) allele and that the levels of intracellular cyclic AMP (cAMP) were also elevated, suggesting that Crg2 also negatively regulates the Gpa1-cAMP signaling pathway. We further showed that Crg2 interacted with Gpa3 and Gpa1, but not Gpa2, in a pulldown assay and that Crg2 maintained a higher in vitro GTPase-activating protein activity toward Gpa3 and Gpa1 than to Gpa2. Finally, we found that dysregulation of cAMP due to the Crg2 mutation attenuated virulence in a murine model of cryptococcosis. Taken together, our study reveals Crg2 as an RGS (regulator of G-protein signaling) protein of multiregulatory function, including one that controls mating distinctly from Crg1 and one that serves as a novel inhibitor of Gpa1-cAMP signaling.

scholarly journals Multiple RGS proteins alter neural G protein signaling to allow C. elegans to rapidly change behavior when fed

2000 ◽  
Vol 14 (16) ◽  
pp. 2003-2014 ◽  
Author(s):  
Meng-Qiu Dong ◽  
Daniel Chase ◽  
Georgia A. Patikoglou ◽  
Michael R. Koelle
Keyword(s):  
G Protein ◽  
Egg Laying ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
C Elegans ◽  
Change Behavior ◽  
Rgs Protein ◽  
Heterotrimeric G Protein Signaling

Regulators of G protein signaling (RGS proteins) inhibit heterotrimeric G protein signaling by activating G protein GTPase activity. Many mammalian RGS proteins are expressed in the brain and can act in vitro on the neural G protein Go, but the biological purpose of this multiplicity of regulators is not clear. We have analyzed all 13 RGS genes in Caenorhabditis elegans and found that three of them influence the aspect of egg-laying behavior controlled by Go signaling. A previously studied RGS protein, EGL-10, affects egg laying under all conditions tested. The other two RGS proteins, RGS-1 and RGS-2, act as Go GTPase activators in vitro but, unlike EGL-10, they do not strongly affect egg laying when worms are allowed to feed constantly. However, rgs-1; rgs-2double mutants fail to rapidly induce egg-laying behavior when refed after starvation. Thus EGL-10 sets baseline levels of signaling, while RGS-1 and RGS-2 appear to redundantly alter signaling to cause appropriate behavioral responses to food.

scholarly journals In vitro selection of proteins with desired characteristics using mRNA-display

Methods ◽  
2013 ◽  
Vol 60 (1) ◽  
pp. 55-69 ◽  
Author(s):  
C. Alexander Valencia ◽  
Jianwei Zou ◽  
Rihe Liu
Keyword(s):  
In Vitro Selection ◽  
Mrna Display ◽  
Selection Of
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