CDB15:0000066 APLN — APLNR

ABSTRACT: Apelin peptides have recently been identified to be the endogenous ligands for the G protein-coupled receptor APJ. However, little is known about the physiological roles of this ligand-receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure-activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C-terminal apelin peptide, apelin-13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre-proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT-PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co-localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.

ABSTRACT: The APJ receptor is widely expressed in the human central nervous system (CNS). Apelin was recently identified as the endogenous peptidic ligand for human APJ. Studies with animal models suggested that APJ and apelin play an important role in the hypothalamic regulation of water intake and the endocrine axis, in the regulation of blood pressure, and in cardiac contractility. Apelin has been found to block the activity of APJ as a human immunodeficiency virus type I (HIV-1) coreceptor. In this study, we combined chemical synthetic approaches with alanine substitution to evaluate the structural requirements for interactions with the APJ receptor. We demonstrated that apelin peptides in aqueous solution adopt a random conformation, and the positive charge and hydrophobic residues of apelin-13 play important roles in interactions with the APJ receptor. We have observed an important correlation between receptor binding affinity and cell-cell fusion inhibitory activity. The elucidation of structural requirements of apelin-13 in its interaction with the APJ receptor is critical for further investigation of apelin-APJ functions in vivo and in the design of small molecular inhibitors for potential treatment of HIV-1 infection in the CNS.

ABSTRACT: Apelin is a novel neuropeptide involved in the regulation of body fluid homeostasis and cardiovascular functions. It acts through a G protein-coupled receptor, the APJ receptor. We studied the structure-activity relationships of apelin at the rat apelin receptor, tagged at its C-terminal end with enhanced green fluorescent protein and stably expressed in CHO cells. We evaluated the potency of N- and C-terminal deleted fragments of K17F to bind with high affinity to the apelin receptor, and to inhibit cAMP production and to induce apelin receptor internalization. We first characterized the internalization and trafficking of the rat apelin receptor. This receptor was internalized via a clathrin-dependent mechanism and our results suggest that receptor trafficking may follow a recycling pathway. We then tried to identify the amino acids of K17F required for apelin activity. The first five N-terminal and the last two C-terminal amino acids of K17F were not essential for apelin binding or the inhibition of cAMP production. However, the full-length sequence of K17F was the most potent inducer of apelin receptor internalization because successive N-terminal amino-acid deletions progressively reduced internalization and the removal of a single amino acid at the C-terminus abolished this process. Finally, the most novel observation of this work is that hypotensive actions of apelin peptides correlate best with the ability of those ligands to internalize. Thus, apelin receptor signaling and endocytosis are functionally dissociated, possibly reflecting the existence of several conformational states of this receptor, stabilized by the binding of different apelin fragments to the apelin receptor.

ABSTRACT: In the search for an endogenous ligand of the orphan G protein-coupled receptor APJ, the presence of the ligand in various tissue extracts was examined by measuring the increase in extracellular acidification rate of the cells expressing the APJ receptor as a specific signal induced by the interaction of the receptor and ligand. By monitoring this activity, we isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The structures of bovine and human apelin preproproteins were deduced from the sequences of the corresponding cDNAs. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions. Synthetic peptides derived from the C-terminal amino acid sequence of bovine preproapelin were capable of specifically promoting the acidification rate in the cells expressing the APJ receptor in a range from 10(-7) to 10(-10) M, indicating that apelin is an endogenous ligand for the APJ receptor.