Differences in the C-terminus contribute to variations in trafficking between rat and human 5-HT(2A) receptor isoforms: identification of a primate-specific tripeptide ASK motif that confers GRK-2 and beta arrestin-2 interactions.
Internalization and recycling of G-protein coupled receptors are important cellular processes regulating receptor function. These are receptor-subtype and cell type-specific. Although important, trafficking variations between receptor isoforms of different species has received limited attention. We report here, differences in internalization and recycling between rat and human serotonin 2A receptor (5-HT(2A)R) isoforms expressed in human embryonic kidney 293 cells in response to serotonin. Although the human and rat 5-HT(2A)Rs differ by only a few amino acids, the human receptor takes longer to recycle to the cell surface after internalization, with the additional involvement of beta arrestin-2 and G-protein receptor kinase 2. The interaction of beta arrestin-2 with the human receptor causes the delay in recycling and is dependent on a primate-specific ASK motif present in the C-terminus of the receptor. Conversion of this motif to NCT, the corresponding sequence present in the rat isoform, results in the human isoform trafficking like the rat receptor. Replacing the serine 457 with alanine in the ASK motif of human isoform resulted in faster recycling, although with continued arrestin-dependent internalization. This study establishes significant differences between the two isoforms with important implications in our understanding of the human 5-HT(2A)R functions; and indicates that extrapolating results from non-human receptor isoforms to human subtypes is not without caveats.
Bhattacharya
Aditi
A
National Centre for Biological Sciences, Tata Institute of Fundamental Research, UAS-GKVK Campus, Bangalore 560065, Karnataka, India.
Sankar
Shobhana
S
Panicker
Mitradas M
MM
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D004705:Endocytosis / Q000235:genetics; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D016296:Mutagenesis / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D044402:Receptor, Serotonin, 5-HT2A / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D018709:Statistics, Nonparametric; D014162:Transfection / Q000379:methods; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; C000605257:Arrb2 protein, rat; D019390:Arrestins; D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; D020033:Protein Isoforms; D044402:Receptor, Serotonin, 5-HT2A; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012694:Serine; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2