Apelin binding to human APJ receptor leads to biased signaling.
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.



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.



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 mutant183MDYS186-AAAA in ECL2 showed Gi-biased signaling while268KTL270-AAA in ECL3 showed Gqbiasing. C281A mutant in ECL3 was deficient in all assays.



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.



This work explains the importance of extracellular loop domains in ligand binding, receptor activation and downstream signaling of human APJR.
Signal Transduction Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India.
Prashant
P
Kumar
Signal Transduction Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India.
Anisha
A
Ashokan
Signal Transduction Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India. Electronic address: agk@iitm.ac.in.
Gopala Krishna
GK
Aradhyam
D019943:Amino Acid Substitution; D000818:Animals; D000073861:Apelin / Q000235:genetics / Q000378:metabolism*; D000075244:Apelin Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D017124:Conserved Sequence; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000071557:beta-Arrestins / Q000378:metabolism
C115436:APLN protein, human; C104575:APLNR protein, human; D000073861:Apelin; D000075244:Apelin Receptors; D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; C495199:apelin-13 peptide; D000071557:beta-Arrestins
APJ receptor; Apelin; G(q) protein; Intra-cellular Ca(2+) rise; β-arrestin