Structure-Function Analysis of Immune Receptor AtRLP23 with Its Ligand nlp20 and Coreceptors AtSOBIR1 and AtBAK1

Pattern-triggered immunity is an inherent feature of the plant immune system. Recognition of either microbe-derived surface structures (patterns) or of plant materials released due to the deleterious impact of microbial infection is brought about by plasma membrane pattern recognition receptors (PRRs). PRRs composed of leucine-rich repeat (LRR) ectodomains are thought to mediate sensing of proteinaceous patterns and to initiate signaling cascades culminating in the activation of generic plant defenses. In contrast to LRR receptor kinases, LRR receptor proteins (LRR-RPs) lack a cytoplasmic kinase domain for initiation of downstream signal transduction. LRR-RPs form heteromeric constitutive, ligand-independent complexes with coreceptor SOBIR1. Upon ligand binding to LRR-RPs, recruitment of coreceptor SERK3/BAK1 results in formation of a ternary PRR complex. Structure-function analysis of LRR-RP-type PRRs is missing. AtRLP23 constitutes an LRR-RP PRR that mediates recognition of a peptide motif (nlp20) found in numerous bacterial, fungal, and oomycete necrosis and ethylene-inducing peptide 1-like proteins (NLPs). We here report the use of a series of AtRLP23 variants to decipher subdomains required for ligand binding and interaction with coreceptors AtSOBIR1 and AtBAK1, respectively. Deletion of LRR1 or LRR3 subdomains efficiently abrogated the ability of AtRLP23 receptor variants to confer nlp20 pattern sensitivity, to bind nlp20, and to recruit AtBAK1 into a ternary PRR complex. This suggests that the very N-terminal part of the AtRLP23 ectodomain is crucial for receptor function. Deletion of the intracellular 17-amino-acid tail of AtRLP23 reduced but did not abolish receptor function, suggesting an auxiliary role of this domain in receptor function. We further found that interaction of AtRLP23 and other LRR-RP-type PRRs with AtSOBIR1 does not require the AtRLP23 LRR ectodomain but is brought about by a GxxxG protein dimerization motif in the transmembrane domain and a stretch of negatively charged glutamic acid residues in the outer juxtamembrane domain of the receptor. Further, AtRLP23 levels were found to be unaltered in Atsobir1-1 mutant genotypes, suggesting that AtSOBIR1 does not act as a protein scaffold in stabilizing LRR-RP-type PRRs in Arabidopsis.

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Ligand Binding and G-protein Coupling of the Serotonin1A Receptor in Cholesterol-enriched Hippocampal Membranes

Bioscience Reports ◽

10.1007/s10540-006-9009-9 ◽

2006 ◽

Vol 26 (2) ◽

pp. 79-87 ◽

Cited By ~ 3

Author(s):

Amitabha Chattopadhyay ◽

Md. Jafurulla ◽

Thomas J. Pucadyil

Keyword(s):

Ligand Binding ◽

G Protein ◽

Protein Interactions ◽

Transmembrane Domain ◽

Function Analysis ◽

Membrane Lipid ◽

Receptor Function ◽

Serotonin1a Receptor ◽

Significant Difference ◽

Lipid Environment

The serotonin1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Since a large portion of such transmembrane receptors remains in contact with the membrane lipid environment, lipid–protein interactions assume importance in the structure-function analysis of such receptors. We have earlier reported the requirement of cholesterol for serotonin1A receptor function in native hippocampal membranes by specific depletion of cholesterol using methyl- β-cyclodextrin. In this paper, we monitored the serotonin1A receptor function in membranes that are enriched in cholesterol using a complex prepared from cholesterol and methyl-β-cyclodextrin. Our results indicate that ligand binding and receptor/G-protein interaction of the serotonin1A receptor do not exhibit significant difference in native and cholesterol-enriched hippocampal membranes indicating that further enrichment of cholesterol has little functional consequence on the serotonin1A receptor function. These results therefore provide new information on the effect of cholesterol enrichment on the hippocampal serotonin1A receptor function.

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Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)82073-7 ◽

1993 ◽

Vol 268 (15) ◽

pp. 10920-10926 ◽

Cited By ~ 2

Author(s):

S. Tanaka ◽

K. Saito ◽

J.C. Reed

Keyword(s):

Structure Function ◽

Cell Survival ◽

Transmembrane Domain ◽

Function Analysis

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High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719900400105 ◽

1999 ◽

Vol 4 (1) ◽

pp. 27-32 ◽

Cited By ~ 36

Author(s):

Ilona Kariv ◽

Michelle E. Stevens ◽

Davette L. Behrens ◽

Kevin R. Oldenburg

Keyword(s):

Ligand Binding ◽

G Protein ◽

High Throughput ◽

High Throughput Screening ◽

Transmembrane Domain ◽

Receptor Function ◽

Camp Production ◽

Ligand Interaction ◽

G Protein Coupled ◽

Native Ligand

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

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A novel uniquely ovary-expressed insulin-like receptor in the female prawn, Macrobrachium rosenbergii (Decapoda, Palaemonidae)

Crustaceana ◽

10.1163/15685403-bja10096 ◽

2021 ◽

Vol 94 (3) ◽

pp. 263-281

Author(s):

Hai-Jing Xu ◽

Jian-Wen Li ◽

Yi-Lai Chen ◽

Jin-Shu Yang ◽

Wen-Ming Ma ◽

...

Keyword(s):

Tyrosine Kinase ◽

Ligand Binding ◽

Transmembrane Domain ◽

Macrobrachium Rosenbergii ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Distribution Analysis ◽

Female Reproduction ◽

Separate Branch ◽

Insulin Signalling Pathway

Abstract The insulin signalling pathway is one of the most studied pathways, including ovary maturation and female reproduction. However, downstream elements involved in this cascade remain unknown. Here, we identified and characterized an insulin-like receptor (IR) in female Macrobrachium rosenbergii, and named as Mro-IR. The deduced Mro-IR contained conserved domains of IR proteins, including two ligand-binding domains, a furin-like domain, two fibronectin-3 domains, a transmembrane domain and an intracellular tyrosine kinase domain in order. Multiple sequence alignment of the first ligand-binding domain and the tyrosine kinase domain in Mro-IR revealed a high degree of similarity to other representative IRs. Mro-IR was clustered into a separate branch with IRs from decapods by phylogenetic analysis. Additionally, tissue distribution analysis showed that Mro-IR was uniquely expressed in the ovary of M. rosenbergii. This study would possibly provide a new highlight to the molecular mechanism of insulin-like signalling cascade, regulating female sexual differentiation in crustaceans.

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A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis

Blood ◽

10.1182/blood-2009-08-236976 ◽

2010 ◽

Vol 115 (2) ◽

pp. 363-369 ◽

Cited By ~ 48

Author(s):

Andrew D. Mumford ◽

Ban B. Dawood ◽

Martina E. Daly ◽

Sherina L. Murden ◽

Michael D. Williams ◽

...

Keyword(s):

Ligand Binding ◽

Chinese Hamster Ovary ◽

Transmembrane Domain ◽

Chinese Hamster ◽

Thromboxane A2 ◽

Receptor Function ◽

Platelet Dysfunction ◽

Structural Element ◽

Thromboxane A2 Receptor

AbstractWe investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A2 receptor (TxA2R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA2R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA2R, U46619 did not increase cytosolic free Ca2+ concentration, indicating loss of receptor function. The TxA2R antagonist [3H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA2R. This is the second naturally occurring TxA2R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA2R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA2R function in vivo.

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