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.
Highlights
Replacement of the secretin receptor amino terminus with that ofthe vasoactive intestinal polypeptide (VIP) receptor resulted in biological responsiveness typical of the VIP receptor (EC50 = 120 11M for secretin, 1.7 11M for VIP)
We have demonstrated that 1) the extracellular amino terminus of the VIP receptor is the critical domain responsible for the selectivity of activation by VIP; 2) the same domain of the secretin receptor is important for the selectivity of high affinity binding and activation by secretin but is not sufficient by itself to provide this function; 3) the first extracellular loop of the secretin receptor is a key domain for selective agonist binding and activation; and 4) in this series of constructs high affinity secretin binding correlated well with secretin-like biological activity, whereas VIP binding could be dissociated from its biological responses
The secretin receptor was activated by secretin with an EC so of 1.5 nMand by VIP with an EC so of 127 nM
Summary
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 constructed a series of six chimeric receptors, exchanging the amino terminus, the first extracellular loop, or both in secretin and VIP receptors. Secretin and vasoactive intestinal polypeptide (VIP)l receptors belong to a recently described family of G protein-coupled receptors [1,2,3] that are only 12% homologous with other members of this superfamily and lack the signature sequences that.
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