Potent and Protease Resistant Azapeptide Agonists of the GLP-1 and GIP Receptors.

The gut-derived peptide hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important physiological roles. Stabilized agonists of the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) for the management of diabetes and obesity have generated widespread enthusiasm and have become blockbuster drugs. These therapeutics are refractory to the action of dipeptidyl peptidase-4 (DPP4), that catalyzes rapid removal of the two N-terminal residues of the native peptides, in turn severely diminishing their activity profiles.  Here we report that a single atom change from carbon to nitrogen in the backbone of the entire peptide make them refractory to DPP4 action while still retaining full potency and efficacy at their respective receptors.  This was accomplished by use of aza-amino acids, that are bioisosteric replacements for a-amino acids that perturb the structural backbone and local side chain conformations.  Molecular dynamics simulations reveal that aza-amino acid can populate the same conformational space that GLP-1 adopts when bound to the GLP-1R. The insertion of an aza-amino acid at the second position from the N-terminus in semaglutide and in a dual agonist of GLP-1R and GIPR further demonstrates its capability as a viable alternative to current DPP4 resistance strategies while offering additional structural variety.

Mutation-Induced Functional Alterations of CCR6.

The Cys-Cys chemokine receptor 6 (CCR6) is a well-established modulator of inflammation. Although several genetic associations have been identified between CCR6 polymorphisms and immune system disorders (e.g., rheumatoid arthritis and Crohn's disease), the pharmacological effects of naturally occurring missense mutations in this receptor have yet to be characterized. In this study, we initially assessed G protein-mediated signaling and observed that wild-type (WT) CCR6 exhibited ligand-independent activity. In addition, we found that the five most frequent CCR6 missense variants (A89T, A150V, R155W, G345S, and A369V) exhibited decreased basal and/or ligand induced Gαi protein signaling. To complement the study of these loss-of-function variants, we engineered a set of constitutively active CCR6 receptors. Selected mutations enhanced basal G protein-mediated signaling up to 3-fold relative to the WT value. Using a bioluminescence resonance energy transfer assay we investigated the ability of each naturally occurring and engineered CCR6 receptor mutant to recruit ß-arrestin. In contrast to G protein-mediated signaling, ß-arrestin mobilization was largely unperturbed by the naturally occurring loss-of-function CCR6 variants. Elevated recruitment of ß-arrestin was observed in one of the engineered constitutively active mutants (T98P). Our results demonstrate that point mutations in CCR6 can result in either a gain or loss of receptor function. These observations underscore the need to explore how CCR6 natural variants may influence immune cell physiology and human disease.

Novel Probes Establish Mas-Related G Protein-Coupled Receptor X1 Variants as Receptors with Loss or Gain of Function.

The Mas-related G protein-coupled receptor X1 (MrgprX1) is a human seven transmembrane-domain protein with a putative role in nociception and pruritus. This receptor is expressed in dorsal root ganglion neurons and is activated by a variety of endogenous peptides, including bovine adrenal medulla peptide (BAM) and γ2-melanocyte-stimulating hormone (γ2-MSH). In the present work, we study how naturally occurring missense mutations alter the activity of MrgprX1. To characterize selected receptor variants, we initially used the endogenous peptide ligand BAM8-22. In addition, we generated and characterized a panel of novel recombinant and synthetic peptide ligands. Our studies identified a mutation in the second intracellular loop of MrgprX1, R131S, that causes a decrease in both ligand-mediated and constitutive signaling. Another mutation in this region, H133R, results in a gain of function phenotype reflected by an increase in ligand-mediated signaling. Using epitope-tagged variants, we determined that the alterations in basal and ligand-mediated signaling were not explained by changes in receptor expression levels. Our results demonstrate that naturally occurring mutations can alter the pharmacology of MrgprX1. This study provides a theoretical basis for exploring whether MrgprX1 variability underlies differences in somatosensation within human populations.

Prolonged Activation of the GLP-1 Receptor via Covalent Capture.

The incretin gut hormone glucagon-like peptide-1 (GLP-1) has become a household name because of its ability to induce glucose-dependent insulin release with accompanying weight loss in patients. Indeed, derivatives of the peptide exert numerous pleiotropic actions that favorably affect other metabolic functions, and consequently, such compounds are being considered as treatments for a variety of ailments. The ability of native GLP-1 to function as a clinical drug is severely limited because of its short half-life in vivo. All of the beneficial effects of GLP-1 come from its agonism at the cognate receptor, GLP-1R. In our quest for long-lived activation of the receptor, we hypothesized that an agonist that had the ability to covalently cross-link with GLP-1R would prove useful. We here report the structure-guided design of peptide analogues containing an electrophilic warhead that could be covalently captured by a resident native nucleophile on the receptor. The compounds were evaluated using washout experiments, and resistance to such washing serves as an index of prolonged activation and covalent capture, which we use to tabulate longevity and robust long-lived GLP-1R agonism. The addition of SulF (cross-linkable warhead), an N-terminal trifluoroethyl group (for protease protection), and a C18 diacid lipid (protractor) all contributed to the increased wash resistance of GLP-1. The most effective compound based on the wash resistance metric, C2K26DAC18_K34SulF, has all three elements outlined and may serve as a blueprint and a proof-of-concept scaffold for the design of clinically useful molecules.

Molecular design of peptide therapeutics via N-terminal modification.

Glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and glucagon are three naturally occurring peptide hormones that mediate glucoregulation. Several agonists representing appropriately modified native ligands have been developed to maximize metabolic benefits with reduced side-effects and many have entered the clinic as type 2 diabetes and obesity therapeutics. In this work, we describe strategies for improving the stability of the peptide ligands by making them refractory to dipeptidyl peptidase-4 catalyzed hydrolysis and inactivation. We describe a series of alkylations with variations in size, shape, charge, polarity, and stereochemistry that are able to engender full activity at the receptor(s) while simultaneously resisting enzyme-mediated degradation. Utilizing this strategy, we offer a novel method of modulating receptor activity and fine-tuning pharmacology without a change in peptide sequence.

A Robust Platform for the Molecular Design of Potent, Protease-Stable, Long-Acting GIP Analogues.

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid peptide hormone that regulates postprandial glucose levels. GIP binds to its cognate receptor, GIPR, and mediates metabolic physiology by improved insulin sensitivity, ß-cell proliferation, increased energy consumption, and stimulated glucagon secretion. Dipeptidyl peptidase-4 (DPP4) catalyzes the rapid inactivation of GIP within 6 min in vivo. Here, we report a molecular platform for the design of GIP analogues that are refractory to DPP4 action and exhibit differential activation of the receptor, thus offering potentially hundreds of GIP-based compounds to fine-tune pharmacology. The lead compound from our studies, which harbored a combination of N-terminal alkylation and side-chain lipidation, was equipotent and retained full efficacy at GIPR as the native peptide, while being completely refractory toward DPP4, and was resistant to trypsin. The GIP analogue identified from these studies was further evaluated in vivo and is one of the longest-acting GIPR agonists to date.

Naturally occurring HCA1 missense mutations result in loss of function: potential impact on lipid deposition.

The hydroxy-carboxylic acid receptor (HCA1) is a G protein-coupled receptor that is highly expressed on adipocytes and considered a potential target for the treatment of dyslipidemia. In the current study, we investigated the pharmacological properties of naturally occurring variants in this receptor (H43Q, A110V, S172L, and D253H). After transient expression of these receptors into human embryonic kidney 293 cells, basal and ligand-induced signaling were assessed using luciferase reporter gene assays. The A110V, S172L, and D253 variants showed reduced basal activity; the S172L mutant displayed a decrease in potency to the endogenous ligand L-lactate. Both the S172L and D253H variants also showed impaired cell surface expression, which may in part explain the reduced activity of these receptors. The impact of a loss in HCA1 function on lipid accumulation was investigated in the adipocyte cell line, OP9. In these cells, endogenous HCA1 transcript levels rapidly increased and reached maximal levels 3 days after the addition of differentiation media. Knockdown of HCA1 using siRNA resulted in an increase in lipid accumulation as assessed by quantification of Nile Red staining and TLC analysis. Our data suggest that lipid homeostasis may be altered in carriers of selected HCA1 missense variants.

Membrane tethered bursicon constructs as heterodimeric modulators of the Drosophila G protein-coupled receptor rickets.

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, we have explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled receptor rickets (rk). Rk/bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. We initially engineered two distinct MTL constructs, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the α or ß bursicon subunit. Coexpression of the two complementary bursicon MTLs triggered rk-mediated signaling in vitro. We were then able to generate functionally active bursicon MTLs in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-ß or ß-α. Carboxy-terminal deletion of 32 amino acids in the ß-α MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble bursicon. We have thus generated membrane-anchored bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of bursicon in the genetically tractable Drosophila model organism. In addition, our success in generating functionally diverse bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins.

TGF-beta regulates T-cell neurokinin-1 receptor internalization and function.

Substance P (SP) is a proinflammatory mediator implicated in inflammatory bowel disease (IBD) and other inflammatory states. SP acts by stimulating the neurokinin-1 receptor (NK-1R) on T lymphocytes and other cell types, and regulates these cells in a complex interplay with multiple cytokines. The mechanisms of interaction among these inflammatory mediators are not yet fully understood. Here, we demonstrate that function of the NK-1R, a member of the G protein-coupled receptor (GPCR) superfamily, is modulated by TGF-beta. The latter acts not on a GPCR but via serine-threonine kinase-class receptors. By flow confocal image analysis, we demonstrate that TGF-beta delays SP-induced NK-1R internalization on mucosal T cells isolated from a mouse model of IBD and on granuloma T cells in murine schistosomiasis. Furthermore, luciferase reporter-gene assays revealed that NK-1R stimulation activates the nuclear factor of activated T cell- and activator protein-1-dependent signaling pathways, which are known triggers of effector T-cell cytokine production. TGF-beta markedly increases SP-induced activation of these signaling cascades, suggesting that delayed NK-1R internalization results in enhanced signaling. Providing a link to amplified immune function, SP and TGF-beta, when applied in combination, trigger a strong release of the proinflammatory cytokines IFN-gamma and IL17 from intestinal inflammatory T cells, whereas either agonist alone shows no effect. These observations establish precedent that members of two distinct receptor superfamilies can interact via a previously unrecognized mechanism, and reveal a paradigm of GPCR transregulation that is relevant to IBD and possibly other disease processes.

Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function.

Class B1 (secretin family) G protein-coupled receptors (GPCRs) modulate a wide range of physiological functions, including glucose homeostasis, feeding behavior, fat deposition, bone remodeling, and vascular contractility. Endogenous peptide ligands for these GPCRs are of intermediate length (27-44 aa) and include receptor affinity (C-terminal) as well as receptor activation (N-terminal) domains. We have developed a technology in which a peptide ligand tethered to the cell membrane selectively modulates corresponding class B1 GPCR-mediated signaling. The engineered cDNA constructs encode a single protein composed of (i) a transmembrane domain (TMD) with an intracellular C terminus, (ii) a poly(asparagine-glycine) linker extending from the TMD into the extracellular space, and (iii) a class B1 receptor ligand positioned at the N terminus. We demonstrate that membrane-tethered peptides, like corresponding soluble ligands, trigger dose-dependent receptor activation. The broad applicability of this approach is illustrated by experiments using tethered versions of 7 mammalian endogenous class B1 GPCR agonists. In parallel, we carried out mutational studies focused primarily on incretin ligands of the glucagon-like peptide-1 receptor. These experiments suggest that tethered ligand activity is conferred in large part by the N-terminal domain of the peptide hormone. Follow-up studies revealed that interconversion of tethered agonists and antagonists can be achieved with the introduction of selected point mutations. Such complementary receptor modulators provide important new tools for probing receptor structure-function relationships as well as for future studies aimed at dissecting the tissue-specific biological role of a GPCR in vivo (e.g., in the brain vs. in the periphery).

A Non-Perturbative Molecular Grafting Strategy for Stable and Potent Therapeutic Peptide Ligands.

The gut-derived incretin hormone, glucagon-like peptide-1 (GLP1), plays an important physiological role in attenuating post-prandial blood glucose excursions in part by amplifying pancreatic insulin secretion. Native GLP1 is rapidly degraded by the serine protease, dipeptidyl peptidase-4 (DPP4); however, enzyme-resistant analogues of this 30-amino-acid peptide provide an effective therapy for type 2 diabetes (T2D) and can curb obesity via complementary functions in the brain. In addition to its medical relevance, the incretin system provides a fertile arena for exploring how to better separate agonist function at cognate receptors versus susceptibility of peptides to DPP4-induced degradation. We have discovered that novel chemical decorations can make GLP1 and its analogues completely DPP4 resistant while fully preserving GLP1 receptor activity. This strategy is also applicable to other therapeutic ligands, namely, glucose-dependent insulinotropic polypeptide (GIP), glucagon, and glucagon-like peptide-2 (GLP2), targeting the secretin family of receptors. The versatility of the approach offers hundreds of active compounds based on any template that target these receptors. These observations should allow for rapid optimization of pharmacological properties and because the appendages are in a position crucial to receptor stimulation, they proffer the possibility of conferring "biased" signaling and in turn minimizing side effects.

The µ-opioid receptor variant N190K is unresponsive to peptide agonists yet can be rescued by small-molecule drugs.

The µ-opioid receptor (MOR) plays an important role in modulating analgesia, feeding behavior, and a range of autonomic functions. In the current study, we investigated the degree to which 13 naturally occurring missense mutations affect the pharmacological properties of the human MOR. After expression of each receptor in human embryonic kidney 293 cells, signaling (Gα(i/o)-mediated) induced by peptide agonists was assessed using luciferase reporter gene assays. Multiple mutants (S66F, S147C, R260H, R265C, R265H, and S268P) show a significant reduction in agonist potency. At the N190K variant, agonist-mediated signaling was essentially absent. Enzyme-linked immunosorbent assay, microscopic analysis, and radioligand binding assays revealed that this mutant shows markedly reduced cell-surface expression, whereas all other receptor variants were expressed at normal levels. Surface expression of the N190K variant could be increased by incubation with the alkaloid agonist buprenorphine or with either naltrexone or naloxone, structurally related MOR antagonists. We were surprised to find that both putative antagonists, despite being inactive at the wild-type MOR, triggered a concentration-dependent increase in N190K receptor-mediated signaling. In contrast, peptidic ligands failed to promote expression or rescue function of the N190K mutant. Subsequent analysis of the N190K variant in an ethnically diverse cohort identified this isoform in a subgroup of African Americans. Taken together, our studies reveal that the N190K mutation leads to severe functional alterations and, in parallel, changes the response to established MOR ligands. The extent to which this mutation results in physiological abnormalities or affects drug sensitivity in selected populations (e.g., those with chronic pain or addiction) remains to be investigated.

Pharmacological characterization of human incretin receptor missense variants.

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut-derived incretin hormones that regulate blood glucose levels. In addition to their widely accepted insulinotropic role, there is evidence that GLP-1 modulates feeding behavior and GIP regulates lipid metabolism, thereby promoting postprandial fat deposition. In this study, we investigated whether naturally occurring polymorphisms in the GLP-1 receptor (GLP-1R) and the GIP receptor (GIP-R) affect the pharmacological properties of these proteins. After transient expression of the receptors in human embryonic kidney 293 cells, basal and ligand-induced cAMP production were assessed by use of luciferase reporter gene assays. Our data reveal that the wild-type GIP-R displays a considerable degree of ligand-independent activity. In comparison, the GIP-R variants C46S, G198C, R316L, and E354Q show a marked decrease in basal signaling that may, at least in part, be explained by reduced cell surface expression. When stimulated with GIP, the C46S and R316L mutants display significantly reduced potency (>1000 and 25- fold, respectively) compared with wild type. Complementary competition binding assays further demonstrate that the C46S variant fails to bind radio-iodinated GIP, whereas all other GIP-R mutants maintain normal ligand affinity. In contrast to the GIP-R, the wild-type GLP-1R lacks constitutive activity. Furthermore, none of the 10 GLP-1R missense mutations showed an alteration in pharmacological properties versus wild type. The extent to which abnormalities in GIP-R function may lead to physiological changes or affect drug sensitivity in selected populations (e.g., obese, diabetic individuals) remains to be further investigated.

Two naturally occurring mutations in the type 1 melanin-concentrating hormone receptor abolish agonist-induced signaling.

The melanin-concentrating hormone (MCH) receptor type 1 (MCHR1) is a seven-transmembrane domain protein that modulates orexigenic activity of MCH, the corresponding endogenous peptide agonist. MCH antagonists are being explored as a potential treatment for obesity. In the current study, we examined the pharmacological impact of 11 naturally occurring mutations in the human MCHR1. Wild-type and mutant receptors were transiently expressed in human embryonic kidney 293 cells. MCHR1-mediated, Gα(i)-dependent signaling was monitored by using luciferase reporter gene assays. Two mutants, R210H and P377S, failed to respond to MCH. Five other variants showed significant alterations in MCH efficacy, ranging from 44 to 142% of the wild-type value. At each of the MCH-responsive mutants, agonist potency and inhibition by (S)-methyl 3-((3-(4-(3-acetamidophenyl)piperidin-1-yl)propyl)carbamoyl)-4-(3,4-difluorophenyl)-6-(methoxymethyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (SNAP-7941), an established MCHR1 small-molecule antagonist, were similar to wild type. To explore the basis for inactivity of the R210H and P377S mutants, we examined expression levels of these receptors. Assessment by enzyme-linked immunosorbent assay revealed that cell surface expression of both nonfunctional receptors was comparable with wild type. Overnight treatment with SNAP-7941, followed by washout of antagonist, enhanced MCH induced signaling by the wild-type receptor and restored MCH responsiveness of the P377S but not the R210H variant. It is of note that the two loss-of-function mutants were identified in markedly underweight individuals, raising the possibility that a lean phenotype may be linked to deficient MCHR1 signaling. Formal association studies with larger cohorts are needed to explore the extent to which signaling-deficient MCHR1 variants influence the maintenance of body weight.

Pharmacological analysis of human D1 AND D2 dopamine receptor missense variants.

Drugs targeting dopamine receptors have been the focus of much research over the past 30 years, in large part because of their role in treating multiple pathological conditions including Parkinson's disease, schizophrenia, Tourette's syndrome, and hyperprolactinemia. Missense mutations in G protein-coupled receptors (GPCRs) can alter basal and/or ligand-induced signaling, which in turn can affect individuals' susceptibility to disease and/or response to therapeutics. To date, five coding variants in the human D1 receptor (hD1R; T37P, T37R, R50S, S199A, and A229T) and three in the human D2 receptor (hD2R; P310S, S311C, and T351A) have been reported in the NCBI single nucleotide polymorphism database. We utilized site-directed mutagenesis to generate cDNAs encoding these receptor isoforms. After expression in either HEK293 or neuronal GT1 cells, basal and ligand-induced signaling of each of these receptors was determined and compared to wild type. In addition, we investigated expression levels of each recombinant receptor and the effect of inverse agonist administration. Our data demonstrate that naturally occurring amino acid substitutions in the hD1R can lead to alterations in expression levels as well as in basal and ligand-induced signaling. The potency and efficacy of dopamine, synthetic agonists (i.e., fenoldopam, SKF-38393, SKF-82958, and SCH23390), and inverse agonists [i.e., flupenthixol and (+)butaclamol] were reduced at selected hD1R variants. Furthermore, inverse agonist induced effects on expression levels were sensitive to selected amino acid substitutions. In contrast to the hD1R variants, hD2R polymorphisms did not affect ligand function or receptor expression. The observation that the hD1R mutations induce significant alterations in pharmacologic properties may have implications both for disease susceptibility and/or therapeutic response to dopaminergic ligands.

Search for alpha-helical propensity in the receptor-bound conformation of glucagon-like peptide-1.

To elucidate the receptor-bound conformation of glucagon-like peptide-1 (GLP-1), a series of conformationally constrained GLP-1 analogues were synthesized by introducing lactam bridges between Lys(i) and Glu(i)(+4) to form alpha-helices at various positions. The activity and affinity of these analogues to GLP-1 receptors suggested that the receptor-bound conformation comprises two alpha-helical segments between residues 11-21 and 23-34. It is notable that the N-terminal alpha-helix is extended to Thr(11), and that Gly(22) plays a pivotal role in arranging the two alpha-helices. Based on these findings, a highly potent bicyclic GLP-1 analogue was synthesized which is the most conformationally constrained GLP-1 analogue reported to date.

Basal receptor activation by locally produced glucagon-like peptide-1 contributes to maintaining beta-cell function.

Glucagon-like peptide 1 (GLP-1) is a physiological stimulus of pancreatic beta-cell function. This enteroendocrine hormone is produced by intestinal L cells, and is delivered via the bloodstream to GLP-1 receptors (GLP-1Rs) on pancreatic beta-cells. In addition, there is evidence that beta-cell GLP-1Rs maintain sustained basal activity even in the absence of intestinal peptide, an observation that has raised the question whether these receptors have some degree of ligand-independent function. Here, we provide an alternative explanation for basal receptor activity based on our finding that biologically relevant amounts of fully processed GLP-1 are locally generated by insulinoma cell lines, as well as by alpha-cells of isolated rat islets in primary culture. Presence of GLP-1 was established by immunocytochemistry, as well as by selective ELISAs and bioassays of cell supernatants. A GLP-1R antagonist significantly reduced insulin secretion/production in beta-TC-6 insulinoma cells and isolated rat islets, suggesting a functionally important loop between locally produced GLP-1 and its cognate receptor. Treatment with this antagonist also inhibited the growth of beta-TC-6 cells. These observations provide novel insight into the function of insulin-producing cell lines and native beta-cells during in vitro culture, and they support the idea that locally produced GLP-1 may play a role in intra-islet regulation.

A human glucagon-like peptide-1 receptor polymorphism results in reduced agonist responsiveness.

Glucagon-like peptide-1 (GLP-1) and its cognate receptor play an important physiological role in maintaining blood glucose homeostasis. A GLP-1 receptor (GLP-1R) polymorphism in which threonine 149 is substituted with a methionine residue has been recently identified in a patient with type 2 diabetes but was not found in non-diabetic control subjects. We have functionally assessed the recombinant GLP-1R variant after transient expression in COS-7 and HEK 293 cells. Compared to the wild type receptor, the variant GLP-1R showed (i) similar expression levels, (ii) 60-and 5-fold reduced binding affinities, respectively, for two GLP-1R full agonists, GLP-1 and exendin-4, and (iii) markedly decreased potencies of these peptides in triggering cAMP-mediated signaling (despite conserved efficacies). In contrast to full agonists, the efficacy of the primary GLP-1 metabolite/GLP-1R partial agonist, GLP-1 (9-36) amide, was essentially abolished by the T149M substitution. By hydropathy analysis, the polymorphism localizes to transmembrane domain 1, suggesting this receptor segment as a novel determinant of agonist affinity/efficacy. These findings reveal that naturally occurring sequence variability of the GLP-1R within the human population can result in substantial loss-of-function. A genetic link between the T149M variant and increased susceptibility to type 2 diabetes remains to be established.

Established theory of radiation-induced decay is not generalizable to Bolton-Hunter labeled peptides.

Peptide hormones radiolabeled with 125I are widely used for the pharmacological characterization of cognate receptors. As a prerequisite for calculating ligand affinities from competition binding assays, and for estimating receptor densities from such studies, it is necessary to know the concentration of bioactive radioligand that is used in respective experiments. It has been demonstrated previously that radioiodinated peptides undergo decay catastrophe, i.e. disintegration of the radioactive label leads to the concomitant destruction of the carrier peptide. Here, we demonstrate that decay catastrophe does not apply to two peptide hormones that are iodinated by Bolton-Hunter conjugation: cholecystokinin octapeptide and glucagon-like peptide 2. The function of aged samples of these radioligands at corresponding recombinantly expressed receptors was assessed by measuring ligand-induced inositol phosphate production or generation of cyclic AMP, respectively. Both of the tested compounds, although predicted by decay catastrophe to contain little or subthreshold remaining bioactivity, stimulated an unexpectedly high level of receptor-mediated second messenger signaling. Quantitative comparison of observed functions with those of corresponding unlabeled peptides suggested that the bioactivity of each radioligand had been largely conserved despite the radioactive decay of the iodine label. Consistent with an apparent absence of decay catastrophe, we noted that the specific radioactivity, when determined immediately following peptide iodination, was close to the theoretical maximum but exponentially decreased over time. These findings raise the possibility that attachment of a Bolton-Hunter conjugate may shield labeled peptides from radiation-induced damage, a scenario that should be considered when performing radioligand binding experiments.

Conserved cholecystokinin receptor transmembrane domain IV amino acids confer peptide affinity.

The Cholecystokinin type 1 and type 2 receptors (CCK-1R and CCK-2R) share >50% amino acid identity, as well as subnanomolar affinity for the endogenous peptide cholecystokinin octapeptide (CCK-8). Although it is likely that these two receptor subtypes share amino acids that confer CCK-8 affinity, it has been difficult to identify such residues. We have examined the role of several transmembrane domain (TMD) IV residues that are common to both CCK receptor subtypes. In both the CCK-1R and CCK-2R, we demonstrate that alanine substitution of two TMD IV residues, which are highly conserved among all known CCK receptor subtypes and species homologs, significantly decrease CCK-8 affinity. Despite the observed decrease in peptide binding, the mutant receptors maintain close to wild-type affinity for the respective subtype selective nonpeptide ligands, 3H-labeled L-364,714 (CCK-1R) and 3H-labeled L-365,260 (CCK-2R), suggesting conserved tertiary structure of these mutants. Assessment of CCK-8-induced inositol phosphate production at each of the mutant CCK receptors revealed normal peptide efficacy. In contrast, peptide potencies are reduced in parallel with the observed decreases in affinity. Taken together, these findings suggest that important peptide affinity determinants are localized on TMD IV, a region that has not previously been considered a major contributor to ligand affinity in either CCK receptors or other G protein-coupled peptide receptors.