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A Gopala Krishna
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A Gopala Krishna
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A Gopala Krishna
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Aradhyam, Gopala K.
Aradhyam, G. K.
Krishna Aradhyam, Gopala
Aradhyam, Gopala Krishna
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3 results
Now showing 1 - 3 of 3
- PublicationCritical APJ receptor residues in extracellular domains that influence effector selectivity(01-11-2021)
;Ashokan, Anisha ;Harisankar, Harikumar Sheela ;Kameswaran, MythiliHuman APJ receptor/apelin receptor (APJR), activated by apelin peptide isoforms, regulates a wide range of physiological processes. The role of extracellular loop (ECL) domain residues of APJR in ligand binding and receptor activation has not been established yet. Based on multiple sequence alignment of APJ receptor from various organisms, we identified conserved residues in the extracellular domains. Alanine substitutions of specific residues were characterized to evaluate their ligand binding efficiency and Gq-, Gi-, and β-arrestin-mediated signaling. Mutation-dependent variation in ligand binding and signaling was observed. W197A in ECL2 and L276L277W279-AAA in ECL3 were deficient in Gi and β-arrestin signaling pathways with relatively preserved Gq-mediated signaling. T169T170-AA, Y182A, and T190A mutants in ECL2 showed impaired β-arrestin-dependent cell signaling while maintaining G protein-mediated signaling. Structural comparison with angiotensin II type I receptor revealed the importance of ECL2 and ECL3 residues in APJR ligand binding and signaling. Our results unequivocally confirm the specific role of these ECL residues in ligand binding and in orchestrating receptor conformations that are involved in preferential/biased signaling functions. - PublicationMolecular determinants on extracellular loop domains that dictate interaction between β-arrestin and human APJ receptor(01-03-2019)
;Ashokan, Anisha ;Kameswaran, MythiliThe human APJ receptor (APJR), activated by apelin isoforms, regulates cardiovascular functions and fluid homeostasis. Understanding its structure-function relationship is crucial for a comprehensive knowledge of signalling aberrations that cause several physiological disorders. Here, we demonstrate the influence of extracellular loop (ECL) domains in the mechanism of β-arrestin-mediated signalling from human APJR: Apelin system. Alanine mutations of evolutionarily conserved residues were characterized using receptor internalization, β-arrestin pull-down, Akt phosphorylation and cell migration assay. C281A and 268KTL270-AAA in ECL3 were deficient in all assays, whereas 183MDYS186-AAAA mutant in ECL2 showed impaired β-arrestin-mediated signalling but demonstrated Gi-dependent cell migration. Our findings establish that conserved residues in the extracellular domain play a prominent role in modulating receptor interactions with the β-arrestin signalling cascade. - PublicationApelin binding to human APJ receptor leads to biased signaling(01-12-2016)
;Kumar, Prashant ;Ashokan, AnishaBackground Human APJ receptor (APJR), a rhodopsin family G-Protein Coupled Receptor (GPCR), activated by isoforms of peptide ligand apelin causing potent inotropic effect, is involved in cardiac function, angiogenesis and maintenance of fluid homeostasis. APJR is expressed in various organs e.g., heart, brain, kidney, muscles, etc. Hence, problems in APJR signaling lead to severe dysregulation in the pathophysiology of an organism. Methods Based on multiple sequence alignment of receptors from various organisms, we observe a large number of conserved residues in the extracellular side. Mutational studies including calcium mobilization, receptor internalization and ERK1/2 phosphorylation assays were performed. Results Stimulation of APJR and its mutants with apelin-13 led to mutation-dependent variation in receptor activation, intracellular Ca2+ rise, and its subsequent downstream signaling. The mutant 183MDYS186-AAAA in ECL2 showed Gi-biased signaling while 268KTL270-AAA in ECL3 showed Gq biasing. C281A mutant in ECL3 was deficient in all assays. Conclusion Conserved residues in the ECL2 of APJR are key for ligand binding, activation mechanism, and selective downstream signaling. Additionally, we demonstrate that Cys281 (in ECL3) mediated disulfide linkage is important for ligand recognition and receptor activation. General significance This work explains the importance of extracellular loop domains in ligand binding, receptor activation and downstream signaling of human APJR.