Cloning, pharmacological characterization and brain distribution of the rat apelin receptor.
The peptide apelin, recently isolated from bovine stomach tissue extracts, has been identified as an endogenous ligand of the human putative receptor protein related to the angiotensin receptor AT(1) (APJ). In this article, we report cloning of the rat apelin receptor cDNA. The sequence shares 90% identity with the human APJ receptor and 31% with the rat AT(1A) angiotensin receptor. Subsequently a stable CHO cell line expressing the receptor fused at its C-terminal part with the enhanced green fluorescent protein (EGFP) was established, allowing to verify its cell surface distribution and to determine the affinity of various apelin and angiotensin fragments on the cloned receptor. As shown for the human APJ receptor, the rat apelin receptor expressed in the cell line was negatively coupled to adenylate cyclase. The apelin fragment K17F (Lys(1)-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe(17)) inhibited forskolin-stimulated cAMP production at sub-nanomolar concentrations whereas angiotensin II and angiotensin III were inactive. N-terminal elongation of K17F with a tyrosine or the N-terminal deletion of the first four amino acids did not modify the inhibitory action of K17F on cAMP production. In contrast, deletion of the first seven amino acids of K17F or substitution of phenylalanine by an alanine residue at the C-terminus completely abolished the activity of the peptide. In situ hybridization analysis of apelin receptor mRNA expression in the adult rat brain showed intense labeling in the hypothalamus, especially in the supraoptic and the paraventricular nuclei. The anterior and intermediate lobes of the pituitary were also highly labeled, as well as the pineal gland. Labeling was also found in extrahypothalamic structures such as the piriform cortex, the nucleus of the lateral olfactory tract, the central grey matter, the pars compacta of the substantia nigra, the dorsal raphe nucleus, the entorhinal cortex, the dentate gyrus and the Ammon's horn. The hypothalamic and hypophyseal distribution of the receptor suggests an involvement of apelin in the control of neuro- and adenohypophyseal hormone release, whereas its presence in the pineal gland and in discrete higher brain structures points out to possible roles in the regulation of circadian rhythms and of water and food intake behavior.
De Mota
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N
Institut National de la Santé et de la Recherche Médicale, Unité 36, Collège de France, Chaire de Médecine Expérimentale, Paris, France.
Lenkei
Z
Z
Llorens-Cortès
C
C
D000595:Amino Acid Sequence; D000809:Angiotensins / Q000494:pharmacology; D000818:Animals; D000073861:Apelin; D000075244:Apelin Receptors; D001483:Base Sequence; D001923:Brain Chemistry / Q000235:genetics*; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism* / Q000494:pharmacology; D002940:Circadian Rhythm / Q000502:physiology; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007032:Hypothalamus, Anterior / Q000737:chemistry / Q000502:physiology; D017403:In Situ Hybridization; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010286:Paraventricular Hypothalamic Nucleus / Q000737:chemistry / Q000502:physiology; D010902:Pituitary Gland / Q000737:chemistry / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011945:Receptors, Angiotensin / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000235:genetics* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D014162:Transfection
C115436:APLN protein, human; C104575:APLNR protein, human; D000809:Angiotensins; D000073861:Apelin; D000075244:Apelin Receptors; C498579:Apln protein, rat; C491284:Aplnr protein, rat; D002352:Carrier Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D011945:Receptors, Angiotensin; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D005576:Colforsin; D000242:Cyclic AMP