CDB15:0001335 SCT — SCTR

ABSTRACT: Secretin and vasoactive intestinal polypeptide (VIP) receptors are closely related G protein-coupled receptors in a recently described family possessing a large amino-terminal ectodomain. We postulated that this domain might be critical for agonist recognition and therefore constructed a series of six chimeric receptors, exchanging the amino terminus, the first extracellular loop, or both in secretin and VIP receptors. Constructs were expressed in COS cells and characterized by cAMP generation and binding of both secretin and VIP radio-ligands. Wild type receptors demonstrated high affinity binding of respective ligands (IC50 values (in nM): at the secretin receptor: 2.2 for secretin, > 1000 for VIP; at the VIP receptor: 2.2 for VIP, > 1000 for secretin) and appropriately sensitive and selective biological responses (EC50 values (in nM): at the secretin receptor: 1.5 for secretin, 127 for VIP; at the VIP receptor: 1.0 for VIP, 273 for secretin). Replacement of the secretin receptor amino terminus with that of the VIP receptor resulted in biological responsiveness typical of the VIP receptor (EC50 = 120 nM for secretin, 1.7 nM for VIP). The converse was not true, with this domain of the secretin receptor not able to provide the same response when incorporated into the VIP receptor (EC50 = 50 nM for VIP, 30 nM for secretin). The addition of both the first loop and the amino terminus of the secretin receptor was effective in yielding a secretin receptor-like response (EC50 = 2.0 nM for secretin, 47 nM for VIP). All chimeric constructs expressing selectivity for secretin-stimulated activity bound this hormone with high affinity (IC50 = 0.2-2.2 nM); however, there was divergence between VIP binding and biological activity. Thus, the amino terminus of secretin and VIP receptors plays a key role in agonist recognition and responsiveness, with the first loop playing a critical complementary role for the secretin receptor.

ABSTRACT: The contribution of the extracellular loops of the secretin receptor to the recognition of secretin was investigated by transfection in CHO cells of chimeric receptors, in which the three loops of the secretin recombinant receptor were replaced by the corresponding sequences of the glucagon receptor. The role of the third loop could not be evaluated as the transfected cells did not respond to secretin. Replacement of extracellular loop 2 reduced markedly the capability of secretin to occupy the receptor but did not alter the capacity of the receptor to discriminate between peptide analogues modified in position 3. Replacement of the first extracellular loop not only reduced the potency of secretin but also decreased the capacity of the receptor to discriminate between ligands having in position 3 an aspartate (as in secretin), an asparagine, or a glutamic acid. This change in receptor properties was reproduced by a single mutation of lysine 173 of the receptor into isoleucine. Thus, the basic amino acid in position 173 is likely to interact with aspartate 3 of secretin. As an aspartate is also present in position 3 of VIP and PACAP, two peptides related to secretin, and a lysine residue is conserved in the first extracellular loop of the VIP and PACAP receptors, this interaction may be a key element of peptide recognition by this receptor family.

ABSTRACT: The biological effects of VIP are mediated by at least two VIP receptors: the VIP1 and the VIP2 receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP1 receptor selective agonsits derived from secretin and GRF. [R16]chicken secretin had IC50 values of binding of 1,10,000, 20, and 3000 nM for the rat VIP1-, VIP2-, secretion- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP1 receptor (IC50 of 60 nM). The chimeric, substituted peptide [K15, R16, L27]VIP(1-7)/GRF(8-27) had IC50 values of binding of 1,10,000, 10,000 and 30,000 nM for the rat VIP1-, VIP2-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC50 of 0.8 nM for the human VIP1 receptor and a low affinity for the human VIP2 receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.

ABSTRACT: Recent evidence indicates that the N-terminal extracellular domain of receptors in the secretin-glucagon receptor family is responsible for ligand recognition. In this report, the N-terminal ectodomain of the human secretin receptor (HSR) was expressed in Escherichia coli, and the ability of this recombinant protein to interact with secretin was investigated by functional assays. The cDNA region encoding the N-terminal ectodomain of HSR linked to the polyhistidine fusion partner was expressed in E. coli. The resulting fusion protein was purified and used for competitive studies. A permanently transfected cell line with the HSR expressed was used in this study. The cell line was able to respond to secretin leading to the elevation of both intracellular cAMP and protein kinase-A activity. Using this cell line, incubation of secretin with the recombinant protein led to a dose-dependent inhibition of both cAMP production and protein kinase-A activity. These findings strongly suggested that the N-terminal ectodomain of HSR alone can act as a functional domain that provides a means to study ligand-receptor interactions of this receptor. The His-tagged recombinant HSR ectodomain may also be used for screening secretin-specific agonists and antagonists by affinity chromatography in the future.