Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.     

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

CRF receptors in the rodent and human cardiovascular systems: species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with ¹²⁵I-[Tyr⁰, Glu¹, Nle¹⁷]-sauvagine or [¹²⁵I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF₂ receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF₂ receptors were detected with ¹²⁵I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with ¹²⁵I-[Tyr⁰, Glu¹, Nle¹⁷]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF₂ receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue -amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 -carbon in the conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.     

The effect of N-terminal substitutions on the biological activity of MSH fragments.

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of -melanotropin, [Glp⁵]-MSH(5–10), [Glp⁵, -Phe⁷]-MSH(5–10), [Sar⁵, -Phe⁷]-MSH(5–10), [Nle⁴, -Phe⁷]-MSH(4–10), [N-carbamoyl]-MSH(5–10), and formyl and acetyl derivatives of -MSH(5–10), [Gly⁵]-MSH(5–10) and [Gly⁵, -Phe⁷]-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.     

Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp⁸⁸, Glu¹⁰⁸, Tyr¹³⁴, Asn¹³⁶, Leu²²⁶ and Gln²²⁷) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp⁸⁸, Tyr¹³⁴ or Asn¹³⁶ eliminated GlcNAc binding activity by GS-II, while those at Glut¹⁰⁸, Leu²²⁶ or Gln²²⁷ did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr¹³⁴ eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu²²⁶ and Gln²²⁷ did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.     

Regional interactions of opioid peptides at μ and δ sites in rat brain

Opiate binding sites in five brain regions were labeled with the μ and δ markers, ³H-morphine and ³H-[D-Ala²,D-leu⁵]enkephalin, respectively. The highest densities of both ³H-morphine and ³H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly ³H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala²,D-leu⁵]enkephalin, β-endorphin and dynorphin(1–13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the μ and δ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the δ binding site and β-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1–13) shows a high affinity and selective preference for the μ binding site over the δ site. The potency of the opioid peptides in displacing the μ and δ markers varies from region to region according to the relative densities of the two opiate binding site populations.     

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.     

Radioimmunoassay for fibroblast growth factor (FGF): release by the bovine anterior pituitary in vitro

A radioimmunoassay (RIA) was developed to measure fibroblast growth factor (FGF) using antiserum generated against a synthetic replicate of [Tyr¹⁰]FGF(1–10). The antisera, previously shown to be capable of inhibiting the biological action of FGF on bovine aortic arch endothelial cells in vitro [1], are highly specific for the amino-terminus of FGF. In the RIA, the antisera recognize the decapeptide antigen [Tyr¹⁰]FGF(1–10) and the intact mitogen on an equimolar basis and show less than 0.01% cross-reactivity with N-acetyl-[Tyr¹⁰]FGF(1–10).

Bovine adenohypophysial cells maintained in primary monolayer culture release and ir-FGF which is indistinguishable from the intact mitogen in as much as it is retained on heparin-Sepharose affinity columns and shows a dose-dependent and parallel displacement in RIA. The release of ir-FGF by the bovine adenohypophysis can be increased with forskolin (10⁻⁵ M) or KCl (50 mM). Preincubation of pituitary cells with 17β-estradiol has no measurable effects on basal ir-FGF, but increases the release after KCl treatment 2–3-fold. These results show that ir-FGF can be released by the bovine adenohypophysis in vitro and lend credence to the hypothesis that FGF plays a physiological role in the homeostatic mechanisms regulating mesoderm-derived cell growth.     

Large and prolonged in vivo response of pancreatic secretion to phenylethylamide and phenylethylester derivatives of Boc-[Nle-Nle]CCK(26–33) in the rat

Supramaximal doses of cholecystokinin (CCK) induce in vitro submaximal biological responses (i.e., smaller by 50% than the response to a maximal dose of CCK), desensitization and residual stimulation, and in vivo secretory inhibition and edematous pancreatitis. It has been reported previously that supramaximal doses of Boc-[Nle²⁸-Nle³¹]CCK(27–32)/-phenylethylester (JMV180) do not produce these effects. The aim of this study was to analyze the in vivo response of pancreatic secretion of the rat to a wide dose range of Boc-[Nle²⁸-Nle³¹]CCK(26–33) (JMV118), an analog of CCK8 with the same activity spectrum as CCK8, to JMV180 and to Boc-[Nle²⁸-Nle³¹]CCK(27–32)-phenylethylamide (JMV170). The three peptides were administered as intravenous infusions and as bolus intravenous injections. In the case of infusions, the same maximal effect was observed with all three peptides. It was obtained with 22.5 pmol/kg · min of JMV118; JMV180 and JMV170 were about 700 times less potent. In the case of bolus injections, the maximal response to JMV118 was observed with 450 pmol/kg, and the response peaked 10–15 min after the injection. Higher doses of JMV118 induced a secretory peak that was smaller and delayed relative to the moment of injection. JMV180 and JMV170 were about 500 times less potent: the maximal response was observed with 218700 pmol/kg and peaked 10–15 min after the injection. Larger doses of JMV180 and JMV170 produced neither supramaximal inhibition nor a delayed peak response, but induced a sustained stimulation of pancreatic secretion that could last more than 3 h after the injection. These data indicate that single large doses of JMV180 and JMV170 can produce a large and long-lasting stimulation of pancreatic secretion in vivo, a goal that cannot be reached with JMV118 or CCK8.     

Probing the opioid receptor complex with (+)-trans-SUPERFIT. II. Evidence that mu ligands are noncompetitive inhibitors of the delta cx opioid peptide binding site.

Previous studies delineated two classes of δ binding sites; a δ binding site not associated with the opioid receptor complex, termed the δ_ncx site, and a δ site associated with the opioid receptor complex, termed the δ_cx site. The δ_ncx site has high affinity for [ -Pen², -Pen⁵]enkephalin, and is synonymous with what is now identified as the δ₁ binding site. Pretreatment of membranes with the δ-selective acylating agents FIT, or (+)-trans-SUPERFIT, deplete membranes of the δ_ncx binding site, which permits the selective labeling of the δ_cx binding site with [³H][ -Ala²,Leu⁵]enkephalin. The present study compared the properties of the δ_cx binding site present in brain membranes pretreated with (+)-trans-SUPERFIT with the properties of the δ_cx site present in untreated membranes. The major findings are: 1) pretreatment of membranes with (+)-trans-SUPERFIT decreased the IC₅₀ values of δ-preferring drugs, and increased the IC₅₀ values of μ-preferring drugs, for the δ_cx binding site; 2) the degree of δ selectivity was highly correlated with the magnitude of the (+)-trans-SUPERFIT-induced shift in the IC₅₀ values; 3) the ligand-selectivity patterns of the μ and δ_cx sites present in (+)-trans-SUPERFIT-pretreated membranes were poorly correlated; 4) whereas μ-preferring drugs were noncompetitive inhibitors of [³H][ -Ala²,Leu⁵]enkephalin binding to the δ_cx site, δ-preferring drugs were competitive inhibitors. Viewed collectively, these data support the hypothesis that the μ and δ_cx binding sites are distinct, provide additional evidence for δ receptor heterogeneity, and suggest that ( (+)-trans-SUPERFIT-pretreated membranes will provide a useful preparation for studying the δ_cx binding site.     

The F1-ATPase beta-subunit is the putative enterostatin receptor

It has been suggested that the F₁-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin_1–7, in the absence and presence of cold enterostatin. ¹²⁵I-β-casomorphin_1–7 weakly binds to the rat F₁-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin_1–7 binding to the F₁-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin_1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin_1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F₁-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F₁-ATPase complex. Western blot analysis showed the F₁-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F₁-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin_1–7 bind to distinct sites on the protein.     

Interaction of calmodulin with synaptic plasma membranes isolated from sheep brain

Calmodulin binding to a membrane fraction enriched in synaptic plasma membranes of sheep brain cells was investigated with [¹²⁵I]calmodulin. Calmodulin binding to these membranes is Ca²⁺-dependent with a half maximal saturation at the pCa value of about 5.5. The binding is reduced by replacing Ca²⁺ with Mg²⁺, but it is significantly enhanced when both cations are present in the medium. Cation-dependent binding is specific and saturable with an apparent K_D of about 47–50 nM and a maximal capacity of about 4 pmol mg⁻¹ protein. The results indicate that synaptic plasma membranes isolated from sheep brain cells interact with calmodulin in a Ca²⁺-dependent, Mg²⁺-facilitated manner.     

Binding of the benzodiazepine ligand [H]-Ro 15–1788 to brain membrane of the saltwater fish Mullus surmuletus

The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [³H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [³H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (K_d value of 2.1 ± 0.2 nM and B_max value of 1400-900 fmol mg⁻¹ of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [³H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [³H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [³H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABA_A receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus.     

High affinity binding of [H]neurotensin to rat uterus

[³H]Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that [³H]NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited [³H]NT binding with the following potencies (IC₅₀): NT 8–13 (0.4 nM), NT 1–13 (4 nM), NT 9–13 (130 nM), NT 1–11, NT 1–8 (>100 μM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.     

Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [¹²⁵I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K⁺-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca²⁺-channel activator BAYK-8644, and blocked K⁺- and BAYK-8644-evoked rise in the intracellular Ca²⁺ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca²⁺ channel-mediated Ca²⁺ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.     

Structure-activity studies with fragments and analogues of salmonid melanin-concentrating hormone

A number of cyclic and linear fragments and analogues of MCH were synthesized and their biological potencies tested using the isolated carp scale melanophore assay. In this system, the cyclic portion MCH(5–14) exhibited only 0.1% bioactivity, which was markedly enhanced by the addition of the exocyclic sequences MCH(15–17) and MCH(1–4). The exocyclic sequence itself, MCH(1–4, 15–17), had minimal activity, however. Substitution of Tyr¹¹ with phenylalanine reduced the potency of the ring structure MCH(5–14) by about 4-fold. Substitution of Gly⁸ with D-alanine reduced the potency of MCH(5–14) 16-fold, while both substitutions together caused a still more marked reduction (200-fold) in bioactivity. Linearized fragments of MCH, extending from MCH(15–17) to [Cys(Acm)^5,14]MCH(1–17), showed a progressive increase in potency. The linearized forms of MCH, MCH(5–17) and MCH(5–14), were approximately 100-fold or less potent than their cyclic forms. The significant increases in bioactivity produced by the addition of the C- and N-terminal exocyclic sequence even to these linearized forms further emphasizes the importance of these regions for interaction at the receptor site.     

Characterization of amylin binding sites in a human hepatoblastoma cell line

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ¹²⁵I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (K_d = 0.11 ± 0.04 nM) and low (K_d = 1.3 ± 0.4 μM) affinity binding sites for ¹²⁵I-amylin in HepG2 cells. The dissociation experiments also showed that ¹²⁵I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ¹²⁵I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace ¹²⁵I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ¹²⁵I[Tyr⁰]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of ¹²⁵I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.