In this report, the first amphibian glucagon receptor (GluR) cDNA was characterized from the liver of the frog Rana tigrina rugulosa. Functional expression of the frog GluR in CHO and COS-7 cells showed a high specificity of the receptor towards human glucagon with an EC 50 value of 0.8 þ 0.5 nM. The binding of radioiodinated human glucagon to GluR was displaced in a dose-dependent manner only with human glucagon and its antagonist (des-His 1 -[Nle 9 -Ala 11 -Ala 16 ]) with IC 50 values of 12.0 þ 3.0 and 7.8 þ 1.0 nM, respectively. The frog GluR did not display any affinity towards fish and human GLP-1s, and towards glucagon peptides derived from two species of teleost fishes (goldfish, zebrafish). These fish glucagons contain substitutions in several key residues that were previously shown to be critical for the binding of human glucagon to its receptor. By RT-PCR, mRNA transcripts of frog GluR were located in the liver, brain, small intestine and colon. These results demonstrate a conservation of the functional characteristics of the GluRs in frog and mammalian species and provide a framework for a better understanding of the molecular evolution of the GluR and its physiological function in vertebrates.z 1999 Federation of European Biochemical Societies.

Peptide histidine isoleucine (PHI), peptide histidine valine (PHV), and vasoactive intestinal polypeptide (VIP) are cosynthesized from the same precursor and share high levels of structural similarities with overlapping biological functions. In this study, the first PHI/PHV receptor was isolated and characterized in goldfish. To study this receptor using homologous peptides, we have also characterized the goldfish prepro-PHI/VIP, and, surprisingly, a shorter transcript lacking the VIP coding region was isolated. A PHI/VIP precursor without the VIP coding sequence has never before been reported. Initial functional expression of the PHI/PHV receptor in Chinese hamster ovary cells revealed that it could be activated by human PHV [50% effective concentration (EC(50)): 43 nM] and to a lesser extent human PHI (EC(50): 133 nM) and helodermin (EC(50): 166 nM) but not fish and mammalian pituitary adenylate cyclase-activating polypeptides and VIPs. Subsequent studies indicated that, similar to the pituitary adenylate cyclase-activating polypeptide receptors (PAC1-R, VPAC1-R, and VPAC2-R), the receptor isolated in this study is able to interact with goldfish PHI and its C-terminally extended form, PHV with EC(50) values 93 and 43 nM, respectively. Northern blot and RT-PCR/Southern blot analyses revealed that the PHI/VIP gene is expressed in the intestine, brain, and gall bladder and the PHI/PHV receptor gene is primarily expressed in the pituitary and to a lesser extend in the intestine and gall bladder, suggesting that PHI/PHV may play a role, notably in the regulation of pituitary function. In conclusion, our results demonstrate for the first time the existence of a PHI/PHV receptor, indicating that the functions of PHI and PHV could be mediated by their own receptor in addition to VIP receptors.

Human secretin receptor is a G protein-coupled receptor that is functionally linked to the cAMP second messenger system by stimulation of adenylate cyclase. To functionally characterize the receptor and evaluate its signal transduction pathway, the full-length human secretin receptor cDNA was subcloned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidification rate was measured by the Cytosensor microphysiometer. Human secretin and biotinylated human secretin were equipotent in both assays in a dose-dependent manner. The EC50 values of stimulating the intracellular cAMP accumulation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmented by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicating that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the protein kinase A inhibitor H-89, suggesting that protein kinase A plays an essential role in the intracellular signaling of the receptor. Upon repeated stimulation by the ligand, the peak acidification responses did not change significantly at both physiological (0.03 nM and 3 nM) and pharmacological (0.3 microM) concentrations of human secretin, suggesting that the human secretin receptor did not exhibit robust homologous desensitization.

In this study, a mutagenesis-based strategy was employed to assess the roles of two highly conserved motifs (KLR and RLAR) within the third endoloop of the human secretin receptor. Block deletion of KLRT and mutation of Lys323 (K 323 I) significantly reduced cAMP accumulation, and these mutations did not affect ligand interaction and receptor number expressed on the cell surface. Thus, the KLRT region at the N terminus of the third endoloop, particularly Lys323, is important for G protein coupling. For the RLAR motif, receptors with substitutions at positions 339 and 342 from Arg to Ala (R 339, 342 A), Glu (R 339, 342 E), or Ile (R 339, 342 I) as well as block deletion of the RLAR motif were all found to be defective in both secretin-binding and cAMP production. Interestingly, a single mutation at the corresponding positions of Arg339 or Arg342 responded as the wild-type human secretin receptor in all functional assays, indicating that the presence of one Arg at either position within the RLAR motif is sufficient for a normal receptor function. Immunofluorescent staining of these mutant receptors showed that these Arg residues are responsible for surface presentation and/or receptor stability. (Endocrinology 142: 3926 -3934, 2001)T HE SECRETIN RECEPTOR has a high affinity for secretin and a relatively low affinity for VIP (1) and belongs to the class II G protein-coupled receptor (GPCR) subfamily. This secretin/VIP receptor subfamily also includes receptors for glucagon, glucagon-like peptide-1 (GLP-1), gastric inhibitory peptide, PTH, pituitary adenylate cyclase activating polypeptide, and GHRH. Generally, the signal transduction mechanism of GPCRs involves ligandinduced changes that affect the conformation of the intracellular surface of the receptors and hence promote the coupling to G proteins (2-6). In the case of the secretin/VIP receptor subfamily, this stimulation process activates adenylate cyclase and eventually leads to the elevation of intracellular cAMP (7-11). Besides cAMP, other intracellular second messengers, such as Ca 2ϩ and inositol phosphates have also been reported (12)(13)(14).Like other GPCRs, the secretin receptor displays a common structural profile in which seven transmembrane domains are linked by alternative exo-and endoloops. Within the same class, there are many conserved amino acid residues, including six well-conserved cysteine residues in the N-terminal extracellular domain and multiple consensus N-glycosylation sites. Nevertheless, these receptors share only 25-50% of amino acid identity among themselves. When compared with other classes of receptors, such as the rhodopsin/␤-adrenergic receptor family, the secretin receptor family is distinct with respect to the primary sequence. Even with this minimal level of sequence homology, comparisons of receptor properties within the family can provide insight into the importance of specific structural domains or motifs. For example, the diversity of the N termini of these receptors in amino acid composition suggests that the N-terminal ...