Critical Contributions of Amino-terminal Extracellular Domains in Agonist Binding and Activation of Secretin and Vasoactive Intestinal Polypeptide Receptors. STUDIES OF CHIMERIC RECEPTORS

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.

Identification of Two Pairs of Spatially Approximated Residues within the Carboxyl Terminus of Secretin and Its Receptor

“…The theme is emerging that aminoterminal regions of these peptides contain key determinants for receptor selectivity, whereas carboxyl-terminal regions contain determinants for high affinity binding (8,9). Of note, the carboxyl-terminal regions of some of the members of this family may even be interchanged while maintaining high affinity binding (9). This feature supports the likely general relevance of the observations in the current work to the entire class II family.…”

“…Chemical reduction and treatment with sulfhydryl-reactive compounds have had substantial negative impact on these receptors (10 -12). Truncation, site-directed mutagenesis, and chimeric receptor studies have also supported the critical role of this domain in the function of this receptor family (9,13).In the present work, we have extended our understanding of the siting of secretin residue 22 when bound to its receptor by defining a receptor residue adjacent to it. We have also established spatial approximation between another residue closer to the carboxyl terminus of secretin (in position 26 of this 27-residue peptide) and a residue within this receptor.…”

“…Chemical reduction and treatment with sulfhydryl-reactive compounds have had substantial negative impact on these receptors (10 -12). Truncation, site-directed mutagenesis, and chimeric receptor studies have also supported the critical role of this domain in the function of this receptor family (9,13).…”

“…The natural agonist ligands of these receptors have helical domains in both carboxyl-terminal and amino-terminal regions (5,6) and have binding determinants that are spread throughout their entire length (3,4,7). The theme is emerging that aminoterminal regions of these peptides contain key determinants for receptor selectivity, whereas carboxyl-terminal regions contain determinants for high affinity binding (8,9). Of note, the carboxyl-terminal regions of some of the members of this family may even be interchanged while maintaining high affinity binding (9).…”

Secretin Receptor Oligomers Form Intracellularly during Maturation through Receptor Core Domains

“…The cells were maintained in a humidified chamber with 5% (v/v) CO 2 at 37 °C and were passaged twice per week on Corning (Acton, MA) tissue culture flasks. Cells inoculated to 10-cm Petri dishes were transfected according to a modified diethylaminoethyl (DEAE)-dextran method (35,37,38), and then were cultured for 24 h in DMEM with serum before lifting with 0.05% (w/v) trypsin and inoculating well plates or flasks for subsequent analyses. All transfections received the same amount of plasmid DNA regardless of the downstream experiment.…”