Selected melanocortin 1 receptor single-nucleotide polymorphisms differentially alter multiple signaling pathways.

The melanocortin 1 receptor (MC1R) is a highly polymorphic G protein-coupled receptor, which is known to modulate pigmentation and inflammation. In the current study, we investigated the pharmacological effects of select single-nucleotide polymorphisms (SNPs) (V60L, R163Q, and F196L). After transient expression of MC1Rs in human embryonic kidney 293 cells, basal and ligand-induced cAMP signaling and mitogen-activated protein kinase (MAPK) activation were assessed by using luciferase reporter gene assays and Western blot analysis, respectively. All receptor variants showed decreased basal cAMP activity. With the V60L and F196L variants, the decrease in constitutive activity was attributable, at least in part, to a reduction in surface expression. The F196L variant also displayed a significant reduction in potency for both the peptide agonist α-melanocyte-stimulating hormone (α-MSH) and the small-molecule agonist 1-[1-(3-methyl-L-histidyl-O-methyl-D-tyrosyl)-4-phenyl-4-piperidinyl]-1-butanone (BMS-470539). In MAPK signaling assays, the F196L variant showed decreased phospho-extracellular signal-regulated kinase levels after stimulation with either α-MSH or BMS-470539. In contrast, the R163Q variant displayed a selective loss of α-MSH-induced MAPK activation; whereas responsiveness to the small-molecule agonist BMS-470539 was preserved. Further assessment of MC1R variants in A549 cells, an in vitro model of inflammation, revealed an enhanced inflammatory response resulting from expression of the F196L variant (versus the wild-type MC1R). This alteration in function was restored by treatment with BMS-470539. Overall, these studies illustrate novel signaling profiles linked to distinct MC1R SNPs. Furthermore, our investigations highlight the potential for small-molecule drugs to rescue the function of MC1R variants that show reduced basal and/or α-MSH stimulated activity.

Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease.

Erythrocytes from patients with homozygous hemoglobin C disease (CC cells) contain less K, Na, and water than do erythrocytes from normal subjects that contain only hemoglobin A (AA cells). In this paper, we provide evidence that the reduced K content and volume of CC cells are due to the activity in these but not in AA cells of a K transport system that is: (a) insensitive to ouabain and bumetanide, and (b) stimulated by increased cell volume, and dependent on internal pH (pHi). When the cation and water content of CC cells was increased (by making the membrane temporarily permeable to cations with nystatin) and the cells were then incubated in an isotonic medium containing 140 mM NaCl and 4 mM KCl, they lost K and shrunk back toward the original volume. This regulatory K and volume decrease was not inhibited by ouabain or bumetanide. When CC cells were incubated in a hypotonic medium, with ouabain and bumetanide, they also lost K and shrunk toward the original volume. This behavior was not observed in control AA cells. The ouabain- and bumetanide-resistant K efflux from CC cells was volume and pH dependent: K efflux from CC cells rose from 5-6 to 20-25 mmol/liter of cells X h, when cell volume was increased by increasing cell solute content (nystatin method) or by exposure to hypotonic media. In CC cells, the dependence of K efflux on pHo had a bell shape, with a maximal flux (20-25 mmol/liter of cells X h) at pHo 6.8-7.0. In contrast, the K efflux from control cells was minimal at pH 7.4 (1.2 mmol/liter of cells X h) and was slightly stimulated by both acid and alkaline pH. In order to study the effect of pHi and pHo on K efflux, CC cells were incubated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM) and acetazolamide (1 mM) at different pHi (6.7, 7.3, and 7.8), and resuspended in media with different pHo (6.75, 7.4, and 8): K efflux was stimulated by reducing pHi but was independent of pHo. The ouabain- and bumetanide-resistant K efflux from CC cells was not inhibited by some inhibitors of the Ca2+-activated K permeability. It seems likely that the genetically determined change in the primary structure of hemoglobin C directly or indirectly causes this modification in K transport. One possible mechanism could involve an electrostatic interaction between C hemoglobin and components of the erythrocyte membrane.

Properties of the Na+-K+ pump in human red cells with increased number of pump sites.

We studied the Na+/K+ pump in red cells from an obese human subject (MAJ) in which the number of pumps/cell was 10-20 times higher than normal. Through measurements of the kinetic properties of several modes of operation of the Na+/K+ pump we determined that the pumps in MAJ cells are kinetically normal. In the presence of adequate metabolic substrate the maximum rates of Na+ pumping and lactate production saturated at 60 and 12 nmol/1 cell per h, respectively. Under physiological conditions pump and "leak" Na+ fluxes were similar in MAJ and normal cells. Since internal Na+ was lower in MAJ than in normal cells (Nai+ approximately 2 and 8 mmol/1 cell, respectively), we conclude that the reduction in cell Na+ allows the Na+/K+ pump in MAJ cells to operate at lower fraction of maximum capacity and to compensate for the increased number of pumps.

The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight.

Food intake and body weight are determined by a complex interaction of regulatory pathways. To elucidate the contribution of the endogenous peptide cholecystokinin, mice lacking functional cholecystokinin-A receptors were generated by targeted gene disruption. To explore the role of the cholecystokinin-A receptor in mediating satiety, food intake of cholecystokinin-A receptor-/- mice was compared with the corresponding intakes of wild-type animals and mice lacking the other known cholecystokinin receptor subtype, cholecystokinin-B/gastrin. Intraperitoneal administration of cholecystokinin failed to decrease food intake in mice lacking cholecystokinin-A receptors. In contrast, cholecystokinin diminished food intake by up to 90% in wild-type and cholecystokinin-B/gastrin receptor-/- mice. Together, these findings indicate that cholecystokinin-induced inhibition of food intake is mediated by the cholecystokinin-A receptor. To explore the long-term consequences of either cholecystokinin-A or cholecystokinin-B/gastrin receptor absence, body weight as a function of age was compared between freely fed wild-type and mutant animals. Both cholecystokinin-A and cholecystokinin-B/gastrin receptor-/- mice maintained normal body weight well into adult life. In addition, each of the two receptor-/- strains had normal pancreatic morphology and were normoglycemic. Our results suggest that although cholecystokinin plays a role in the short-term inhibition of food intake, this pathway is not essential for the long-term maintenance of body weight.

Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene.

It is well established that the gene encoding the hormone secretin is expressed in a specific enteroendocrine cell, the S cell. We now show that the secretin gene is transiently expressed in insulin-producing B cells of the developing pancreatic islets in addition to the intestine. Furthermore, secretin is produced by most established islet cell lines. In order to identify and characterize the regulatory elements within the secretin gene that control tissue-specific expression, we have introduced secretin reporter gene constructions into the secretin-producing HIT and STC-1 cell lines as well as the nonexpressing INR1-G9 glucagonoma line. Analysis of deletion mutants revealed that sequences between 174 and 53 bp upstream from the transcriptional start site are required for maximal expression in secretin-producing cells. This positive element functioned independently of position and orientation. Further deletions into the enhancer resulted in a stepwise loss of transcriptional activity, suggesting the presence of several discrete control elements. The sequence CAGCTG within the secretin enhancer closely resembles that of the core of the B-cell-specific enhancer in the insulin gene. Point mutations introduced into this putative element led to greater than 85% reduction in transcriptional activity. Gel mobility shift assays suggested that a factor in B cells closely related or identical to proteins that bind to the insulin enhancer interacts with the CAGCTG motif in the secretin gene.

CCK receptor polymorphisms: an illustration of emerging themes in pharmacogenomics.

Polymorphisms in G-protein-coupled receptors can alter drug affinity and/or activity. In addition, genetic differences in amino acid sequences can induce ligand-independent signaling, which in turn can lead to disease. With growing efforts in the field of pharmacogenomics, it is anticipated that polymorphism-induced alterations in drug and/or receptor function will be a focus of increasing concern during the course of future drug-development efforts. In this review, the spectrum of pharmacological consequences that result from polymorphisms in the cholecystokinin CCK2 receptor will be discussed, thereby illustrating emerging themes in pharmacogenomics.

Targeted disruption of the murine CCK1 receptor gene reduces intestinal lipid-induced feedback inhibition of gastric function.

Cholecystokinin (CCK), acting at CCK1 receptors (CCK1Rs) on intestinal vagal afferent terminals, has been implicated in the control of gastrointestinal function and food intake. Using CCK1R(-/-) mice, we tested the hypothesis that lipid-induced activation of the vagal afferent pathway and intestinal feedback of gastric function is CCK1R dependent. In anesthetized CCK1R(+/+) ("wild type") mice, meal-stimulated gastric acid secretion was inhibited by intestinal lipid infusion; this was abolished in CCK1R(-/-) mice. Gastric emptying of whole egg, measured by nuclear scintigraphy in awake mice, was significantly faster in CCK1R(-/-) than CCK1R(+/+) mice. Gastric emptying of chow was significantly slowed in response to administration of CCK-8 (22 pmol) in CCK1R(+/+) but not CCK1R(-/-) mice. Activation of the vagal afferent pathway was measured by immunohistochemical localization of Fos protein in the nucleus of the solitary tract (NTS; a region where vagal afferents terminate). CCK-8 (22 pmol ip) increased neuronal Fos expression in the NTS of fasted CCK1R(+/+) mice; CCK-induced Fos expression was reduced by 97% in CCK1R(-/-) compared with CCK1R(+/+) mice. Intralipid (0.2 ml of 20% Intralipid and 0.04 g lipid), but not saline, gavage increased Fos expression in the NTS of fasted CCK1R(+/+) mice; lipid-induced Fos expression was decreased by 47% in CCK1R(-/-) compared with CCK1R(+/+)mice. We conclude that intestinal lipid activates the vagal afferent pathway, decreases gastric acid secretion, and delays gastric emptying via a CCK1R-dependent mechanism. Thus, despite a relatively normal phenotype, intestinal feedback in response to lipid is severely impaired in these mice.

Secretin: structure of the precursor and tissue distribution of the mRNA.

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. The unusually high number of serine, leucine, and arginine residues in secretin has precluded the use of oligonucleotides to screen cDNA libraries to isolate a secretin cDNA. In the present study, a short cDNA encoding porcine secretin was amplified from duodenal mucosal first-strand cDNA template by using 16,384- and 4096-fold degenerate primers in the DNA polymerase chain reaction. From the sequence of the amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a cDNA library. Here we report the sequences of cDNAs encoding the porcine and rat secretin precursors. The predicted amino acid sequences reveal that each precursor consists of a signal peptide, an N-terminal peptide, secretin, and a 72-amino acid C-terminal peptide. Secretin has been highly conserved through evolution. Rat secretin differs from its porcine counterpart by a single glutamine-for-arginine substitution at position 14. In contrast, the amino acid sequences of the C-terminal peptides are only 39% conserved between the two species, suggesting that the C-terminal peptide does not have an essential physiologic function. RNA blot hybridizations reveal that the rat secretin gene is expressed throughout the small intestine. Although secretin immunoreactivity has been localized in the central nervous system by some laboratories, we are unable to detect secretin mRNA in tissues of the central nervous system by Northern blot hybridization.

Species differences between rat and mouse CCKA receptors determine the divergent acinar cell response to the cholecystokinin analog JMV-180.

The cholecystokinin (CCK) analog JMV-180 acts as a partial agonist in rats and a full agonist in mice. Whether this functional variability is due to species differences in CCK receptor structure or to alterations in the cellular environment is unknown. To address this question, an adenoviral construct encoding the rat CCK(A) receptor (AdCCK(A)R) was used to express the rat receptor in acini from CCK(A) receptor-deficient mice (CCK(A)R -/-). Infection of CCK(A)R -/- acini in vitro with pAdCCK(A)R led to a time-dependent increase in (125)I-CCK(8) binding. The affinity for JMV-180 of the adenovirally transferred rat and the endogenous mouse CCK(A) receptors was not different. In native mouse acini, JMV-180 acted as a full agonist (both stimulation and inhibition of amylase release). In contrast, in mouse acini expressing pAdCCK(A)R JMV-180 acted as a partial agonist (only stimulation of amylase release). In addition, the pattern of protein synthesis induced by JMV-180 in CCK(A)R -/- mouse acini infected with AdCCK(A)R resembled the pattern observed in wild-type rats (lack of inhibition) rather than the respective pattern in wild-type mice (inhibition). These data suggest that species differences in the CCK(A) receptor of rats and mice account for the observed divergence in the acinar cell response to JMV-180.

Different splice site utilization generates diversity between the rat and human pancreatic polypeptide precursors.

Pancreatic polypeptide is derived from a polyprotein precursor molecule. Although the amino acid sequences specifying the signal peptide and pancreatic polypeptide are well conserved between the rat and the human, the carboxy-terminal amino acid sequences of the precursors are highly divergent. To better understand the molecular basis of the divergence between the rat and human C-terminal peptides, we have determined the nucleotide sequence of the rat pancreatic polypeptide gene. A comparison between the primary structures of the rat and human genes reveals that the heterogeneity of the C-terminal peptides can be explained in large part by a frameshift mutation and the utilization of an alternative splice donor site in the third exon of the rat gene. As a consequence of the displaced splice site, part of the third exon of the rate gene is homologous to the sequence in the third intron of the human gene. Our results suggest that the rat and human pancreatic polypeptide genes arose from a common ancestral gene, and that differences in the C-terminal domains of the precursor reflect less strict evolutionary constraints than those imposed upon the amino-terminal domains of the precursor.

Minor modifications of a cholecystokinin-B/gastrin receptor non-peptide antagonist confer a broad spectrum of functional properties.

The development of non-peptide agonists for peptide hormone receptors would markedly expand the treatment options for a large number of diseases. However, difficulty in identifying non-peptide molecules which possess intrinsic activity has been a major obstacle in achieving this goal. At present, most of the known non-peptide ligands for peptide hormone receptors appear in standard functional assays to be antagonists. Here, we report that a constitutively active mutant of the human cholecystokinin-B/gastrin receptor, Leu325 --> Glu, offers the potential to detect even trace agonist activity of ligands which, at the wild type receptor isoform, appear to lack efficacy. The enhanced functional sensitivity of the mutant receptor enabled us to detect intrinsic activity of L-365,260, an established non-peptide antagonist for the cholecystokinin-B/gastrin receptor. Extending from this observation, we were able to demonstrate that minor structural modifications could convert L-365, 260 into either: (i) an agonist or (ii) an inverse agonist (attenuates ligand-independent signaling). The ability to confer functional activity to small non-peptide ligands suggests that the properties of endogenous peptide hormones can be mimicked, and even extended, by considerably less complex molecules.

Electrolyte composition and equilibrium in hemoglobin CC red blood cells.

The red cells of two patients with homozygous hemoglobin C disease (CC) were found to have reduced cation and water content when compared to the red cells of two normal subjects that contained only hemoglobin A (AA). The reduction in cation content was of such a magnitude that the intracellular chloride and hydroxyl (and proton) concentrations were within normal limits despite a measured reduction in the concentration of impermeant negative anions in CC as compared with AA cells of 40 mEq/kg of dry cell solids. The osmotic coefficient of hemoglobin C in CC cells was found to be similar to that observed for hemoglobin A in AA cells. We found no evidence for increased amounts of bound or osmotically abnormal water in CC cells. The reduction in cell cation content in CC cells is mainly due to a reduction in cell K content. The reduced K content is probably related to an increased ouabain- and furosemide-insensitive pathway for K transport in CC cells.

The role of the cholecystokinin-B/gastrin receptor transmembrane domains in determining affinity for subtype-selective ligands.

We have examined the role of transmembrane domain amino acids in conferring subtype-selective ligand affinity to the human cholecystokinin-B (CCK-B)/gastrin receptor. Fifty-eight residues were sequentially replaced by the corresponding amino acids from the pharmacologically distinct CCK-A receptor subtype. 125I-CCK-8 competition binding experiments were performed to compare all mutant CCK-B/gastrin receptor constructs with the wild type control. Affinities for the nonselective agonist, CCK-8, as well as the subtype-selective peptide (gastrin), peptide-derived (PD135,158), and nonpeptide (L365,260) and L364,718) ligands were assessed. All of the mutants retained relatively high affinity for CCK-8, suggesting that the tertiary structure of these receptors was well maintained. Only eight of the amino acid substitutions had a significant effect on subtype selective binding. When compared with the wild type, single point mutations in the CCK-B/gastrin receptor decreased affinity for gastrin, L365,260, and PD135,158 up to 17-,23-, and 61-fold, respectively. In contrast, the affinity for L364,718 increased up to 63-fold. None of the single amino acid substitutions, however, was sufficient to fully account for the subtype selectivity of any tested compound. Rather, CCK-B/gastrin receptor affinity appears to be influenced by multiple residues acting in concert. The 8 pharmacologically important amino acids cluster in the portion of the transmembrane domains adjacent to the cell surface. The spatial orientation of these residues was analyzed with a rhodopsin-based three-dimensional model of G-protein coupled receptor structure (Baldwin, J.M. (1993) EMBO J. 12, 1693-1703). This model predicts that the 8 crucial residues project into a putative ligand pocket, similar to the one which is well established for biogenic amine receptors (Caron, M. G., and Lefkowitz, R.J. (1993) Recent Prog. Horm. Res. 48, 277-290; Strader, C.D., Sigal, I.S., and Dixon, R.A. (1989) Trends Pharmacol. Sci. 10, Dec. Suppl., 26-30).

Inter- and intraspecies polymorphisms in the cholecystokinin-B/gastrin receptor alter drug efficacy.

The brain cholecystokinin-B/gastrin receptor (CCK-BR) is a major target for drug development because of its postulated role in modulating anxiety, memory, and the perception of pain. Drug discovery efforts have resulted in the identification of small synthetic molecules that can selectively activate this receptor subtype. These drugs include the peptide-derived compound PD135,158 as well as the nonpeptide benzodiazepine-based ligand, L-740,093 (S enantiomer). We now report that the maximal level of receptor-mediated second messenger signaling that can be achieved by these compounds (drug efficacy) markedly differs among species homologs of the CCK-BR. Further analysis reveals that the observed differences in drug efficacy are in large part explained by single or double aliphatic amino acid substitutions between respective species homologs. This interspecies variability in ligand efficacy introduces the possibility of species differences in receptor-mediated function, an important consideration when selecting animal models for preclinical drug testing. The finding that even single amino acid substitutions can significantly affect drug efficacy prompted us to examine ligand-induced signaling by a known naturally occurring human CCK-BR variant (glutamic acid replaced by lysine in position 288; 288E --> K). When examined using the 288E --> K receptor, the efficacies of both PD135,158 and L-740, 093 (S) were markedly increased compared with values obtained with the wild-type human protein. These observations suggest that functional variability resulting from human receptor polymorphisms may contribute to interindividual differences in drug effects.

Small synthetic ligands of the cholecystokinin-B/gastrin receptor can mimic the function of endogenous peptide hormones.

The gastric cholecystokinin-B/gastrin receptor (CCK-BR) is a key regulator of enterochromaffin-like cell function and proliferation. Over the last decade, a number of small non-peptide CCK-BR "antagonists" have been discovered. Here, we demonstrate that some of these non-peptide ligands in fact possess significant ability to activate the human CCK-BR, and are, therefore, more properly categorized as partial agonists. When tested in COS-7 cells transiently expressing the recombinant human CCK-BR, saturating concentrations of the small "peptoid" ligands PD 135,158 and PD 136,450 stimulated inositol phosphate formation to 23 and 43 percent, respectively, of the maximum response induced by a considerably larger endogenous peptide agonist, cholecystokinin octapeptide. In contrast, the benzodiazepine-derived CCK-BR ligand, YM022, acted as a "true" high-affinity antagonist of cholecystokinin-induced inositol phosphate formation (pA2 = 9.69). Consistent with recent findings in animal experiments, our data reveal that small synthetic ligands have the potential to function as either CCK-BR agonists or antagonists. These dual properties of synthetic molecules must be considered when evaluating candidate drugs for human disease.

Abnormal gastric morphology and function in CCK-B/gastrin receptor-deficient mice.

Mice lacking the cholecystokinin (CCK)-B/gastrin receptor have been generated by targeted gene disruption. The roles of this receptor in controlling gastric acid secretion and gastric mucosal growth have been assessed. The analysis of homozygous mutant mice vs. wild type included measurement of basal gastric pH, plasma gastrin concentrations as well as quantification of gastric mucosal cell types by immunohistochemistry. Mutant mice exhibited a marked increase in basal gastric pH (from 3.2 to 5.2) and about a 10-fold elevation in circulating carboxyamidated gastrin compared with wild-type controls. Histologic analysis revealed a decrease in both parietal and enterochromaffin-like (ECL) cells, thus explaining the reduction in acid output. Consistent with the elevation in circulating gastrin, antral gastrin cells were increased in number while somatostatin cells were decreased. These data support the importance of the CCK-B/gastrin receptor in maintaining the normal cellular composition and function of the gastric mucosa.

A single amino acid of the cholecystokinin-B/gastrin receptor determines specificity for non-peptide antagonists.

The brain cholecystokinin-B/gastrin receptor (CCK-B/gastrin) has been implicated in mediating anxiety, panic attacks, satiety, and the perception of pain. The canine and human CCK-B/gastrin receptors share 90% amino-acid identity and have similar agonist affinities. These receptors can be selectively blocked by the non-peptide benzodiazepine-based antagonists L365260 (ref. 8) and L364718 (ref. 9); however, the binding of these antagonists to the human and canine receptors differs by up to 20-fold, resulting in a reversal of affinity rank order. Here we report the identification of a single amino acid in the sixth transmembrane domain of the CCK-B/gastrin receptor that corresponds to valine 319 in the human homologue and which is critical in determining the binding affinity for these non-peptide antagonists. We show that it is the variability in the aliphatic side chain of the amino acid in position 319 that confers antagonist specificity. Substitution of valine 319 with a leucine residue decreases the affinity for L365260 20-fold while concomitantly increasing the affinity for L364718. An isoleucine in the same position of the human receptor selectively increases affinity for L364718. Interspecies differences in the aliphatic amino acid occupying this single position selectively affect antagonist affinities without altering the agonist binding profile. We therefore conclude that the residues underlying non-peptide antagonist affinity must differ from those that confer agonist specificity. To our knowledge, these findings are the first example in which a critical antagonist binding determinant for a seven-transmembrane-domain peptide hormone receptor has been identified.

Interspecies polymorphisms confer constitutive activity to the Mastomys cholecystokinin-B/gastrin receptor.

The enteroendocrine hormone, gastrin, exerts trophic effects on the gastric mucosa through the CCK-B/gastrin receptor (CCK-BR). To varying degrees in different species, excess circulating gastrin leads to proliferation of enterochromaffin-like cells and to the development of gastric carcinoid tumors. The African rodent, Mastomys natalensis, is distinguished from other mammals by its propensity toward CCK-BR-mediated growth even in the absence of hypergastrinemia. Here, we report that the Mastomys CCK-BR, when expressed in COS-7 cells, differs from the respective human, canine, and rat receptor homologs by its ability to trigger ligand-independent (i.e., constitutive) inositol phosphate formation. To define the molecular basis of this observation, a series of Mastomys-human chimeric receptors was investigated. Functional characterization of these constructs revealed that a limited segment of the Mastomys CCK-BR, transmembrane domain VI through the C-terminal end, is sufficient to confer constitutive activity to the human protein. Mutagenesis studies within this CCK-BR region defined a combination of three Mastomys amino acids that, when introduced into the human receptor, together conferred a level of ligand-independent signaling comparable with the Mastomys CCK-BR. Complementing prior observations that single point mutations can lead to ligand-independent signaling, our findings suggest that multiple naturally occurring amino acid polymorphisms and/or mutations may together result in an enhanced basal level of receptor activity.