skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Differential regulation of G protein signaling in Arabidopsis through two distinct pathways that internalize AtRGS1
In animals, endocytosis of a seven-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic G protein–mediated signaling, depending on the bias of the receptor or ligand, which determines how much one transduction pathway is used compared to another. InArabidopsis thaliana, GPCRs are not required for G protein–coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the seven-transmembrane protein AtRGS1 modulates G protein signaling through ligand-dependent endocytosis, which initiates derepression of signaling without the involvement of canonical arrestins. Here, we found that endocytosis of AtRGS1 initiated from two separate pools of plasma membrane: sterol-dependent domains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and candidate arrestin-like adaptors. Ligand identity (either the pathogen-associated molecular pattern flg22 or the sugar glucose) determined the origin of AtRGS1 endocytosis. Different trafficking origins and trajectories led to different cellular outcomes. Thus, in this system, compartmentation with its associated signalosome architecture drives biased signaling.  more » « less
Award ID(s):
1713880
PAR ID:
10286080
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
American Association for the Advancement of Science (AAAS)
Date Published:
Journal Name:
Science Signaling
Volume:
14
Issue:
695
ISSN:
1945-0877
Page Range / eLocation ID:
Article No. eabe4090
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis. 
    more » « less
  2. G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (β 2 AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (G s ). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to β 2 AR by G s protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from β 2 AR and the conformational interconversions of G s between closed and open conformations in the NE(+)–β 2 AR–G s ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter G s α subunit (G s α) conformational transitions. Our simulations showed that the interdomain movement and the stacking of G s α α1 and α5 helices are significant for increasing the distance between the G s α and β 2 AR, which may indicate a partial dissociation of G s α The distance increase commences when G s α is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of β 2 AR interacting with G s α, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation. 
    more » « less
  3. Summary The plant hormone abscisic acid (ABA) plays crucial roles in regulation of stress responses and growth modulation. Heterotrimeric G‐proteins are key mediators of ABA responses. Both ABA and G‐proteins have also been implicated in intracellular redox regulation; however, the extent to which reversible protein oxidation manipulates ABA and/or G‐protein signaling remains uncharacterized.To probe the role of reversible protein oxidation in plant stress response and its dependence on G‐proteins, we determined the ABA‐dependent reversible redoxome of wild‐type and Gβ‐protein null mutantagb1of Arabidopsis.We quantified 6891 uniquely oxidized cysteine‐containing peptides, 923 of which show significant changes in oxidation following ABA treatment. The majority of these changes required the presence of G‐proteins. Divergent pathways including primary metabolism, reactive oxygen species response, translation and photosynthesis exhibited both ABA‐ and G‐protein‐dependent redox changes, many of which occurred on proteins not previously linked to them.We report the most comprehensive ABA‐dependent plant redoxome and uncover a complex network of reversible oxidations that allow ABA and G‐proteins to rapidly adjust cellular signaling to adapt to changing environments. Physiological validation of a subset of these observations suggests that functional G‐proteins are required to maintain intracellular redox homeostasis and fully execute plant stress responses. 
    more » « less
  4. In deciphering the global signaling capacity of FERONIA receptor kinase, Liu, Yeh, et al. discovered an extracellular phase separation process driven by FERONIA peptide ligand RALF-cell wall polysaccharide pectin interaction, which leads to cognate and non-cognate receptor clustering and promiscuous endocytosis as a coping mechanism in response to environmental stressors. Highlights Cell surface pectin-RALF1 phase separation recruits FERONIA-LLG1 into condensates RALF induces FERONIA-LLG1-dependent promiscuous receptor clustering and endocytosis RALF1-pectin molecular condensates function as surface sensors for stress signals FERONIA-LLG1-mediated global endocytosis ensures plant resilience under stress 
    more » « less
  5. Abstract Under synchronized conidiation, over 2500 gene products show differential expression, including transcripts for bothbrlAandabaA, which increase steadily over time. In contrast, during wall-stress induced by the echinocandin micafungin, thebrlAtranscript is upregulated while theabaAtranscript is not. In addition, whenmpkA(last protein kinase in the cell wall integrity signaling pathway) is deleted,brlAexpression is not upregulated in response to wall stress. Together, these data imply BrlA may play a role in a cellular stress-response which is independent of the canonical BrlA-mediated conidiation pathway. To test this hypothesis, we performed a genome-wide search and found 332 genes with a putative BrlA response element (BRE) in their promoter region. From this set, we identified 28 genes which were differentially expressed in response to wall-stress, but not during synchronized conidiation. This set included seven gene products whose homologues are involved in transmembrane transport and 14 likely to be involved in secondary metabolite biosynthesis. We selected six of these genes for further examination and find that they all show altered expression behavior in thebrlAdeletion strain. Together, these data support the idea that BrlA plays a role in various biological processes outside asexual development. ImportanceTheAspergillus nidulanstranscription factor BrlA is widely accepted as a master regulator of conidiation. Here, we show that in addition to this function BrlA appears to play a role in responding to cell-wall stress. We note that this has not been observed outsideA. nidulans. Further, BrlA-mediated conidiation is highly conserved acrossAspergillusspecies, so this new functionality is likely relevant in otherAspergilli. We identified several transmembrane transporters that have altered transcriptional responses to cell-wall stress in abrlAdeletion mutant. Based on our observation, together with what is known about thebrlAgene locus’ regulation, we identifybrlAβas the likely intermediary in function ofbrlAin the response to cell-wall stress. 
    more » « less