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In order to get more insight into the mechanism of action of the gastrointestinal peptide, motilin, and its role in human physiology, we aimed at characterizing motilin and motilin receptors. Motilin. Sequence analysis of the motilin precursor from several species indicated that the N- and C-terminal regions of the motilin precursor have evolved at different rates. Sequence analysis of the motilin precursor in brain tissue of rabbit and man and motilin radioimmunoassay on tissue extracts, proved that motilin is a brain-gut peptide. Plasma motilin levels are increased in patients with ulcerative colitis or Crohn's disease. A weak correlation between the motilin genotype and the susceptibility to inflammatory bowel disease was demonstrated. Motilin receptors. Motilin receptors are expressed early postnatally and can be regulated by changes in its plasma level. The pharmacophore of motilin consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. In vivo and in vitro studies showed that in the rabbit and human antrum, smooth muscle and neuronal motilin receptors exist which have different characteristics. In the rabbit duodenum motilin's action depends upon the influx of extra- and intracellular Ca2+. Nevertheless, in primary smooth muscle cultures, Ca2+ influx through L-type Ca2+ channels is the major transduction mechanism. The existence of central motilin receptors was demonstrated by autoradiography. Receptor binding studies allowed the identification of two binding sites. In contrast to antral smooth muscle cells, the response to motilin in the human TE671 medulloblastoma cell line, expressing the motilin receptor, relies on intracellular IP3-sensitive Ca2+ stores. The antibiotic erythromycin-A (EM-A) binds to the motilin receptor and induces contractions with the same regional and species specificity as motilin. This interaction was supported by the discovery of motilin antagonists. Structure activity studies led to the development of more powerful erythromycin derivatives, which lack antibiotic properties and which are now in clinical trial for treatment of hypomotility disorders. Conclusion and perspectives. The physiological role of motilin and its receptors in the brain requires further investigation. Erythromycin and its derivatives act as motilin agonists with clinically useful prokinetic potential. The motilin receptor has recently been cloned and has substantial structural homology with the growth hormone secretagogue receptor. This may not only lead to the further characterization of motilin receptor subtypes and aid the development of safe and selective motilin receptor agonists and antagonists, useful for the treatment of GI disorders, but may also give a new dimension to the role of motilin in human physiology.

Agonist action at receptors is highly specific, affected by the structure of both ligand and receptor. Chimeric constructs of structurally related receptors and/or ligands that have biological differences provide an opportunity to correlate a specific structural domain with function. In this work, we have used a cross-chimeric approach to explore the structural basis for rat secretin and vasoactive intestinal polypeptide action at their closely related secretin and VPAC(1) receptors, belonging to class II of the G protein-coupled receptor superfamily. Multiple domains of both ligands and receptors contributed toward their selectivity, with differing combinations of such domains able to support high-potency interactions. The amino-terminal 15 residues of secretin were most critical for potent stimulation of secretin receptors, whereas either the amino- or carboxyl-terminal halves of vasoactive intestinal polypeptide, when complemented by Lys(15), provided potent stimulation of the VPAC(1) receptor. The amino terminus of the VPAC(1) receptor was most critical for potent response to vasoactive intestinal polypeptide, whereas the amino terminus of the secretin receptor was important, but not adequate, requiring the complementation of an extracellular loop domain for potent response to secretin. Differences in the distribution of these determinants within these receptors provided an opportunity to produce a more "universal" receptor that contained the first extracellular loop of the secretin receptor and the remainder of the VPAC(1) receptor. This cross-chimeric approach should be applied to other members of this receptor family to test the emerging themes and to expand these insights as broadly as possible.

The G-protein-coupled peptide YY (PYY)/neuropeptide Y Y1 receptor (Y1R) subtype is highly expressed in the proliferative zone of human colonic crypt epithelial cells but biochemical and biological support for growth effects have been lacking. Using a model gut epithelial cell system, we have stably expressed the human Y1R in IEC-6 cells and show that the Y1R does couple to mitogen-activated protein kinase (MAPK) phosphorylation and cell growth. This pathway uses pertussis-toxin-sensitive G-proteins and betagamma subunits, inhibited by co-transfected alpha-transducin. The Src-family tyrosine kinase inhibitor PP1, as well as specific inhibition of the epidermal growth factor receptor tyrosine kinase (EGFR TK) by PD153035, also blocks PYY stimulation of MAPK. This pathway further requires protein kinase C with EGFR TK inhibition blocking PYY-induced protein kinase Cepsilon (PKCepsilon) translocation to the cell membrane. Finally, we show that PYY stimulates growth in Y1R-expressing gut epithelial cells that is dependent on EGFR TK activity. These results demonstrate a novel pathway involving G(i)/G(o) protein, EGFR and PKC to activate MAPK. Further, they support a role for PYY and the Y1R in regulating growth in human colonic epithelium.

Aims/hypothesis: Synergism between glucose and cAMP in the stimulation of insulin secretion has been suggested to regulate beta cells. This study assessed the importance of an interaction between glucose and cAMP in the stimulation of insulin secretion from human islet cells by investigating expression and functional activity of receptors recognising glucagon, glucagon-like peptide-1 (7-36)amide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)., Methods: Expression of the glucagon, GLP-1 and GIP receptors in human islets was investigated by northern blots and reverse transcription-polymerase chain reaction analysis. Functional activity of these receptors was assessed by the effects of peptides (agonists and antagonists) on glucose-induced insulin release., Results: Human islet cells express transcripts encoding glucagon, GLP-1 and GIP receptors. Glucose (10 mmol/l) stimulated insulin release 4.5 +/- 0.6-fold over basal (2.5 mmol/l). This glucose effect was amplified by 10 nmol/l GLP-1, GIP or glucagon. It was reduced by 51 +/- 6% in the presence of 1 micromol/l of the glucagon-receptor antagonist des-His1-[Glu9]-glucagon-amide (n = 8; p < 0.05), indicating participation of endogenously released glucagon in the process of glucose-induced insulin release. The glucagon-receptor antagonist also suppressed the potentiation of glucose-induced insulin release by addition of 10 nmol/l glucagon., Conclusion/interpretation: These data suggest that human beta cells express functional glucagon receptors which can, similar to incretin hormone receptors, generate synergistic signals for glucose-induced insulin secretion.

Pancreastatin, a chromogranin A-derived peptide widely distributed throughout the neuroendocrine system, has a general inhibitory effect on endocrine secretion and a counterregulatory effect on insulin action. We have recently described the cross-talk of pancreastatin with insulin signaling in rat hepatoma cells (HTC), where it inhibits insulin action and signaling through the serine phosphorylation of the insulin receptor, thereby impairing tyrosine kinase activity. Here, we have characterized pancreastatin receptors and signaling in HTC cells. The pancreastatin effector systems were studied by determining phospholipase C activity in HTC membranes and mitogen-activated protein kinase (MAPK) phosphorylation activity in HTC cells. Binding studies with radiolabeled pancreastatin showed a population of high affinity binding sites, with a B(max) of 8 fmol/mg protein and a K(d) of 0.6 nM. Moreover, we assessed the coupling of the receptor with a G protein system by inhibiting the binding with guanine nucleotide and by measuring the GTP binding to HTC membranes. We found that pancreastatin receptor was coupled with a G alpha(q/11) protein which activates phospholipase C-beta(1) and phospholipase C-beta(3), in addition to MAPK via both beta gamma and alpha(q/11).

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response was abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. Beta-arrestin-1 transfection also induced a significantly decrease in GIP-stimulated cAMP production, and this effect was greater with cotransfection of both GRK2 and beta-arrestin-1 than with either alone. In betaTC3 cells, expression of GRK2 or beta-arrestin-1 attenuated GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion was not affected. GRK2 and beta-arrestin-1 messenger RNAs were identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells. Overexpression of GRK2 enhanced agonist-induced GIPR phosphorylation, but receptor endocytosis was not affected by cotransfection with GRKs or beta-arrestin-1. These results suggest a potential role for GRK2/beta-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR.

We previously reported that intracisternal (i.c.) injection of peptide YY (PYY) and low doses of thyrotropin-releasing hormone (TRH) or TRH analog, RX 77368, increased the resistance of the gastric mucosa to ethanol injury through vagal pathways in rats. The gastroprotective effect of i.c. injection of PYY/neuropeptide NPY (NPY) agonists with differential in vitro affinity to the Y receptor subtypes was examined in urethane-anesthetized rats. Intragastric administration of ethanol (45%, 5 ml/kg) results in mucosal lesions covering 23+/-2% of the gastric corpus in 1 h. PYY (500 ng, i.c.) significantly reduced ethanol-induced gastric lesions by 52%. [Pro34]PYY (PYY-preferring/Y1/Y5/Y4 subtypes) injected i.c. at 50, 100, 200 or 500 ng, reduced dose dependently gastric lesions to 15.4+/-2.2%, 11.4+/-3.1%, 8.6+/-2.9% and 5.4+/-2.2%, respectively. PYY3-36, (Y2/Y4 subtypes), [Leu31, Pro34]NPY (Y1/Y5), NPY (Y3/Y1/Y5/Y2) and pancreatic polypeptide (PP, Y4) injected i.c. at 500 ng did not influence significantly ethanol-induced gastric lesions. Combined i.c. injection of RX 77368 (1 ng) and Pro34PYY (25 ng), at sub-threshold doses given singly, reduced ethanol-induced gastric injury to 12.9+/-2.3% while RX 77368 (1 ng) plus PYY3-36 (500 ng) or [Leu31, Pro34]NPY (25 ng) had no effect. These findings indicate that i.c. PYY-induced gastric protection against 45% ethanol is mediated by a Y receptor subtype which bears similarity with the putative PYY-preferring receptor and distinct from the currently defined Y1/Y5; in addition, there is a synergistic interaction between activation of this PYY-preferring receptor and i.c. TRH to increase the resistance of the gastric mucosa to injury caused by 45% ethanol.

Secretin is a 27-amino acid long peptide hormone that regulates pancreatic water, bicarbonate, enzymes, and potassium ion secretion. The human secretin receptor (hSR) is a glycoprotein consisting of 440 amino acids, of which there are 5 putative N-linked glycosylation sites at positions Asn72, Asn100, Asn106, Asn128 (N-terminal ectodomain), and Asn291 (second exoloop). Through functional analysis of the hSR-transfected cells cultured in the presence of various glycosylation inhibitors, it was found that tunicamycin and castanospermine were able to significantly reduce the secretin-stimulated cAMP response. On the other hand, the effects of other inhibitors, swainsonine and deoxymannojirimycin, were much lower, suggesting that the high mannose-type carbohydrate side-chain is essential to the expression of a fully functional hSR. The role of individual N-linked glycosylation sites was studied by mutation analysis (Asn to Leu or Ser to Ala) coupled to measurements of cAMP accumulation and extracellular acidification rate. The ED50 values of the wild-type receptor in these two assay systems were 0.25 and 0.11 nM, respectively, and mutation at position 100, 106, or 291 did not affect either the ED50 values or the maximal responses in the two assays. However, the Asn72Leu and Ser74Ala mutations reduced the maximal responses and increased the ED50 values in both assays, suggesting that this site is a true glycosylation signal. This hypothesis was further supported by competitive binding studies, the same mutants were found to be defective in binding with [125I]secretin. To evaluate whether the change in receptor function of the mutants is caused by the change in the process of presenting the receptor to the cell surface, the mutants and the wild-type receptor were tagged with a c-Myc epitope at the C-termini. Using an anti-c-Myc monoclonal antibody and confocal microscopy, all of the mutant receptors were found to be expressed and delivered to the plasma membrane.

Peptide YY (PYY) and neuropeptide Y (NPY) are peptides that coordinate intestinal activities in response to luminal and neuronal signals. In this study, using the rat hybrid small intestinal epithelial cell line, hBRIE 380i cells, we demonstrated that PYY- and NPY-induced rearrangement of actin filaments may be in part through a Y1alpha and/or a nonneuronal Y2 receptor, which were cloned from both the intestinal mucosa and the hBRIE 380i cells. A number of PYY/NPY-responsive genes were also identified by subtractive hybridization of the hBRIE 380i cells in the presence or absence of a 6-h treatment with PYY. Several of these genes coded for proteins associated with the cell cytoskeleton or extracellular matrix. One of these proteins was the transmembrane-4 superfamily protein CD63, previously shown to associate with beta(1)-integrin and implicated in cell adhesion. CD63 immunoreactivity, using antibody to the extracellular domain, was highest in the differentiated cell clusters of the hBRIE 380i cells. The hBRIE 380i cells transfected with antisense CD63 cDNA lost these differentiated clusters. These studies suggest a new role for NPY and PYY in modulating differentiation through cytoskeletal associated proteins.

In the liver, pancreastatin exerts a glycogenolytic effect through interaction with specific receptors, followed by activation of phospholipase C and guanylate cyclase. Pancreastatin receptor seems to be coupled to two different G protein systems: a pertussis toxin-insensitive G protein that mediates activation of phospholipase C, and a pertussis toxin sensitive G protein that mediates the cyclic GMP production. The aim of this study was to identify the specific G protein subtypes coupling pancreastatin receptors in rat liver membranes. GTP binding was determined by using gamma-35S-GTP; specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Activation of G proteins was demonstrated by the incorporation of the photoreactive GTP analogue 8-azido-alpha-32P-GTP into liver membranes and into specific immunoprecipitates of different Galpha subunits from soluble rat liver membranes. Pancreastatin stimulation of rat liver membranes increases the binding of gamma-35S-GTP in a time- and dose-dependent manner. Activation of the soluble receptors still led to the pancreastatin dose-dependent stimulation of gamma-35S-GTP binding. Besides, WGA semipurified receptors also stimulates GTP binding. The binding was inhibited by treatment with anti-Galphaq/11 (85%) and anti-Galphai1,2 (15%) sera, whereas anti-Galphao,i3 serum failed to affect the binding. Finally, pancreastatin stimulates GTP photolabeling of particulate membranes. Moreover, it specifically increased the incorporation of 8-azido-alpha-32P-GTP into Galphaq/11 and Galpha, but not into Galphao,i3 from soluble rat liver membranes. In conclusion, pancreastatin stimulation of rat liver membranes led to the activation of Galphaq/11 and Galphai1,2 proteins. These results suggest that Galphaq/11 and Galphai1,2 may play a functional role in the signaling of pancreastatin receptor by mediating the production of IP3 and cGMP respectively.

The secretin amino terminal residues are essential for high affinity binding to the cognate receptor and for the subsequent activation of adenylate cyclase. It has been already established that two basic residues of the receptor TM 2 are involved in the interaction with aspartate 3 of the ligand. The present work investigated the hypothesis that two conserved tyrosine residues of the TM 1 (Tyrosines 124 and 128) could also participate to the positioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mutated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-type receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity of the receptor for secretin, [Glu3]secretin, [Asn3]secretin and the secretin fragment 2-27, and reduced the intrinsic activity of [Asn3]secretin. Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn3]secretin affinity but only 3-fold that of [Glu3]secretin. Secretin and [Glu3]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly with the [Asp3] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 helices.

The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties.

In the present work, the presence of gastric inhibitory polypeptide (GIP) receptors and their functional role in the adrenal cells of three patients with food-dependent Cushing's syndrome were studied. RT-PCR and in situ hybridization studies demonstrated the presence of GIP receptor in the adrenals of the three patients. The presence of this receptor was also demonstrated in two human fetal adrenals, but not in two normal adult human adrenals or in the adrenals of one patient with nonfood-dependent Cushing's syndrome. Freshly isolated cells from patient adrenals responded in a dose-dependent manner to the steroidogenic action of both ACTH and GIP, whereas cells from normal adrenals responded only to ACTH. Treatment of cultured normal adrenal cells with ACTH, but not with GIP, increased the messenger ribonucleic acid (mRNA) levels of cholesterol side-chain cleavage cytochrome P-450, P450c17, and 3beta-hydroxysteroid dehydrogenase, whereas both hormones enhanced these mRNAs in patients' adrenal cells, although the effects of ACTH were greater than those of GIP. Moreover, pretreatment with ACTH enhanced the steroidogenic responsiveness of both normal and patient adrenal cells, whereas GIP caused homologous desensitization, and this was associated with a marked reduction of GIP receptor mRNA levels, as demonstrated by RT-PCR and in situ hybridization. Finally, both ACTH and GIP inhibited DNA synthesis in one patient's adrenal cells, whereas in normal adrenal cells only ACTH had this effect. In conclusion, the present data demonstrate that ectopic expression of functional GIP receptors is the main cause of food-dependent Cushing's syndrome.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.

The pancreatic polypeptide (PP-fold) family of peptides consists of the endocrine peptides, pancreatic polypeptide (PP) and peptide YY (PYY), and the neuroneally derived peptide, neuropeptide Y (NPY). All three peptides are found in the circulation, with PP found primarily in the pancreas and PYY found principally in the gut. NPY is released into the circulation from neuroneal stores in response to stress. These peptides have broad peripheral actions on a number of organs. Not surprisingly, PYY and PP are believed to play an important role in the function of the gastrointestinal tract while NPY is a potent vasconstrictor and may have effects on the gut through the enteric nervous system. In the brain, NPY has been implicated in anxiety and depression, feeding and obesity, memory retention, neuroneal excitability, endocrine function, and metabolism. Recent advances in the molecular biology of the receptors for these peptides have resulted in the identification of at least six receptor subtypes with varying peptide pharmacology. Compared to other G-protein coupled receptor families, the PP-fold peptide receptors exhibit a relatively low level of sequence identity. Further advances in the development of selective agonists and antagonists for individual receptor subtypes will be needed to understand further their role in physiological function.

We have investigated a potential role of motilin in amplifying the postsynaptic muscarinic responses in the rabbit duodenal smooth muscle cells, using the whole cell variant of patch-clamp technique. Stimulation of motilin receptors by exogenously applied motilin (1 nM) resulted in a large increase in carbachol (CCh)-induced atropine-sensitive cation current (ICCh) at threshold concentrations of CCh (0.3-1 microM) at 30 degrees C. This potentiation was abolished in the presence of a specific blocker of motilin receptor (GM109) and was attenuated with increased concentrations of either motilin or CCh, being virtually absent with maximally effective concentrations of these agonists. Motilin failed to potentiate ICCh when the ambient temperature was reduced to 20 degrees C or if the cation current had been directly activated by internal perfusion with guanosine 5'-O-(3-thiotriphosphate) (50 microM) bypassing the muscarinic receptor. These results suggest that some biochemical processes, such as enzymatic reactions, might be involved in the motilin-induced potentiation and that its site of action might be the muscarinic receptor and/or associated G proteins.

Galanin is a 29-amino-acid neuropeptide that is overexpressed in Alzheimer's disease (AD) and impairs performance on rodent learning and memory tasks. M40, a peptidergic galanin receptor ligand, blocks galanin-induced impairments on delayed non-matching-to-position (DNMTP). The present experiments used the 192IgG-saporin lesion model of AD to evaluate the actions of galanin and M40 on DNMTP when cholinergic transmission was reduced. Hippocampal choline acetyltransferase levels were correlated with DNMTP choice accuracy in lesioned rats. Intracerebroventricular (icv) galanin reduced choice accuracy in both the lesioned and sham groups. M40 alone, either icv or intrahippocampal, did not affect choice accuracy in either group. These results suggest that excess galanin can produce further deficits in DNMTP performance in a lesion model of AD, but blocking endogenous galanin is not sufficient alone to improve performance in lesioned rats.

Peptide YY (PYY) is a potent regulator of intestinal secretion. These studies investigated the role of Y1 and Y2 receptor subtypes in mediating the antisecretory effects of PYY on mucosa-submucosa preparations of rat distal colon. Addition of vasoactive intestinal peptide (VIP) to these tissues resulted in a 140 +/- 18% increase in basal short-circuit current (Isc) and the induction of Cl- secretion. VIP-stimulated increases in Isc were abolished by the addition of each of PYY, (Pro34)-PYY, a Y1 receptor-selective agonist, and PYY-(3-36), an endogenous Y2 receptor-selective ligand. However, when tissue neural transmission was blocked with tetrodotoxin, neither PYY nor its receptor subtype-selective analogs were able to inhibit VIP-stimulated increases in Isc. These results suggest that in the rat distal colon, the antisecretory actions of PYY are mediated through a combination of Y1 and Y2 receptor subtypes or through a novel receptor subtype that is unable to discriminate between (Pro34)-PYY and PYY-(3-36).

Exogenous galanin stimulates feeding when injected into forebrain and hindbrain sites, including the third and fourth ventricles (3V and 4V), amygdala, paraventricular nucleus of the hypothalamus (PVN), and nucleus of the solitary tract (NTS). Because the PVN and NTS border the ventricular space, it is possible that feeding stimulated by injection of galanin at these sites may be caused by the transport of galanin through the ventricular system to a remote site of action. The role of ventricular transport of galanin between the 3V and 4V in galanin-induced feeding was examined in this study. Rats were implanted with two guide cannula assemblies: one dorsal to the mesencephalic aqueduct and the other in the 3V or 4V. Feeding in response to 3V or 4V galanin injection was first measured after sham-occlusion of the aqueduct. Subsequently, flow of cerebrospinal fluid between the forebrain and hindbrain ventricles was acutely interrupted by injection of a silicone grease plug into the mesencephalic aqueduct just before assessment of the feeding response to 4V or 3V galanin injection. Aqueduct occlusion did not alter the feeding induced by either 3V or 4V galanin injection, indicating that galanin terminals in both the diencephalon and hindbrain are involved in control of food intake.

Activated receptors for galanin and norepinephrine, and for several other agonists, inhibit insulin release from pancreatic beta-cells via pertussis toxin-sensitive Gi- and Go-proteins and by acting on at least four cellular mechanisms. These mechanisms include repolarization via activation of the ATP-sensitive potassium (K ATP) channel, inhibition of adenylyl cyclase, and inhibition by unknown mechanism at a "distal" site. For norepinephrine and galanin there is also inhibition of the L-type Ca2+ channel. Consequently, during simultaneous activation by multiple agonists, the effectiveness with which a receptor interacts with the G-proteins will, to some extent, determine the responses. This could have important consequences for the beta-cell. Therefore, the G-protein interactions of two activated receptors, those for norepinephrine and galanin, were compared in the same beta-cell membranes. Measurements were made of the rates of receptor-G-protein interaction (by GTPgammaS binding) and of the rates of turnover of G-proteins (by GTPase activity). A comparison was also made of the ability of norepinephrine and galanin to facilitate ADP ribosylation of the alpha-subunits of Gi and Go by cholera toxin (CTX). Such CTX-induced ADP ribosylation of Gi and Go occurs during G-protein interaction with an activated receptor. By measurement of the number of receptors in the membrane preparation used, the relative effectiveness of the two receptors was assessed. The alpha2-adrenergic receptor was found to be markedly more effective than the galanin receptor in activating G-proteins.

Galanin, a neuroendocrine peptide with a multitude of functions, binds to and acts on specific G-protein coupled receptors. Only one galanin receptor subtype, GalRI, has been cloned so far, although pharmacological evidence suggests the presence of more than one galanin receptor subtype. These receptors mediate via different Gi/Go-proteins the inhibition of adenylyl cyclase, opening of K+-channels and closure of Ca2+-channels. Galanin inhibits secretion of insulin, acetylcholine, serotonin and noradrenaline, while it stimulates prolactin and growth hormone release. Determination of structural components of galanin receptors required for binding of the peptide ligand as carried out recently will facilitate the screening and design of molecules specifically acting on galaninergic systems with therapeutic potential in Alzheimer's disease, feeding disorders, pain and depression.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid peptide produced by K cells of the mammalian proximal small intestine and is a potent stimulant of insulin release in the presence of hyperglycemia. However, its relative physiological importance as a postprandial insulinotropic agent is unknown. Using LGIPR2 cells stably transfected with rat GIP receptor cDNA, GIP (1-42) stimulation of cyclic adenosine monophosphate (cAMP) production was inhibited in a concentration-dependent manner by GIP (7-30)-NH2. Competition binding assays using stably transfected L293 cells demonstrated an IC50 for GIP receptor binding of 7 nmol/liter for GIP (1-42) and 200 nmol/liter for GIP (7-30)-NH2, whereas glucagonlike peptide-1 (GLP-1) binding to its receptor on ++betaTC3 cells was minimally displaced by GIP (7-30)-NH2. In fasted anesthetized rats, GIP (1-42) stimulated insulin release in a concentration-dependent manner, an effect abolished by the concomitant intraperitoneal administration of GIP (7-30)-NH2 (100 nmol/ kg). In contrast, glucose-, GLP-1-, and arginine-stimulated insulin release were not affected by GIP (7-30)-NH2. In separate experiments, GIP (7-30)-NH2 (100 nmol/kg) reduced postprandial insulin release in conscious rats by 72%. It is concluded that GIP (7-30)-NH2 is a GIP-specific receptor antagonist and that GIP plays a dominant role in mediating postprandial insulin release.

GH controls its own secretion through a mechanism involving short-loop feedback regulation of the synthesis and release of GH-releasing hormone (GHRH). GHRH neurons coexpress the peptide galanin, but the functional significance of this coexpression is unknown. In this study, we tested the hypotheses that 1) galanin gene expression in GHRH neurons is regulated by GH and 2) somatostatin (SS) or GHRH neurons are a target for the action of galanin in the hypothalamus. First, we compared levels of galanin messenger RNA (mRNA) in GHRH neurons between normal male rats and Lewis dwarf rats, which have markedly reduced blood levels of GH. The brains of normal and dwarf animals were processed for detection of galanin mRNA and GHRH mRNA by double-label in situ hybridization. We observed that Lewis dwarf rats had significantly reduced levels of galanin mRNA in their GHRH neurons (P < 0.05). Next, we tested the hypothesis that GH regulates galanin gene expression in GHRH neurons by experimentally altering circulating levels of GH. Three groups of adult male rats were used: 1) intact rats (n = 7); 2) hypophysectomized (hypox) rats (n = 7); and 3) hypox rats treated with 1.5 mg of rat GH (rGH) over a 3-day period (n = 6). At the end of the treatment period, the animals were killed, and their brains were collected and processed for double-label in situ hybridization for GHRH mRNA and galanin mRNA. The signal level of galanin mRNA in GHRH neurons was reduced in hypox animals to less than 10% of that in intact controls (P < 0.0001); whereas, the levels of galanin mRNA signal in GHRH neurons did not differ significantly between the groups of intact and rGH-treated hypox rats. Finally, to determine whether SS or GHRH neurons are targets for galanin, we used double-label in situ hybridization to determine whether either of these populations of neurons express galanin receptor mRNA. A subset of SS neurons in the PeN appeared to express the galanin receptor mRNA, whereas few, if any, GHRH neurons appeared to do so. We conclude that galanin, like its cotransmitter GHRH, is a target for GH action, and we infer that galanin may play a role in the feedback control of GH secretion by exerting a direct effect on SS neurons.

Gastric inhibitory polypeptide (GIP) potently stimulates insulin secretion from pancreatic islets in the presence of glucose as an incretin. Because the insulinotropic effect of GIP is reduced in NIDDM, it should be clarified whether defects in the GIP receptor gene contribute to the impaired insulin secretion in NIDDM. Using genomic DNA samples from Japanese NIDDM and non-NIDDM subjects, we have investigated the entire coding region of the GIP receptor gene by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). We have identified two missense mutations, Gly198-->Cys (Gly198Cys) in exon 7 and Glu354-->Gln (Glu354Gln) in exon 12. Investigation of the function of GIP receptor with either of these mutations reveals a half-maximal stimulation value of GIP-induced cAMP response in Chinese hamster ovary cells expressing the GIP receptor with Gly198Cys of 6.3 +/- 1.2 x 10(-10) mol/l (n = 3), which was considerably higher than that of the normal GIP receptor, 9.4 +/- 3.8 x 10(-12) mol/l GIP (n = 3), whereas that of the GIP receptor with Glu354Gln was not significantly different from that of the normal GIP receptor. To assess the possible role of the GIP receptor gene in genetic susceptibility to NIDDM, we have examined the allelic frequencies of Gly198Cys and Glu354Gln in NIDDM and control subjects. Association studies show no relationship between NIDDM and either of the two mutations.

Noradrenergic neurons of the locus coeruleus (LC) negatively regulate the endogenous rhythmicity of growth hormone (GH) secretion. These neurons express high concentrations of receptors for somatostatin (SRIH) and galanin (GAL), two neuropeptides which can affect electrical activity of LC neurons and also centrally modulate plasma GH levels. We thus investigated whether somatostatin and galanin receptors located in the LC are involved in GH regulation. Pulsatile patterns of endogenous GH secretion were monitored after unilateral infusion of the peptides into the lateral ventricle (ICV) or into the LC, after lesion of contralateral LC neurons by 6 hydroxydopamine. Neither unilateral LC lesions nor administration of saline affected GH release. When administered ICV, both SRIH (5 micrograms/microliter/15 min) and GAL (1 microgram/microliter/15 min) resulted in a marked increase in GH secretion. Infusion of SRIH into the LC induced a significant but weaker stimulation of plasma GH as compared to ICV injections. In contrast, infusion of GAL into the LC was ineffective. These results indicate that somatostatin can exhibit direct effects on noradrenergic neurons of the LC involved in GH regulation, whereas central effects of galanin on the hormone are mediated by distinct structures.

We studied a 45-yr-old woman with food-dependent Cushing's syndrome. Plasma cortisol levels were subnormal (4-47 nmol/L) after an overnight fast and increased after a mixed meal to values between 500-1000 nmol/L. There was a close correlation between circulating gastric inhibitory polypeptide (GIP) and cortisol levels during normal food intake (r = 0.92; P < 0.0002). Plasma corticotropin (ACTH) levels were undetectable. Nonfasting plasma cortisol levels were not suppressed by low or high doses of dexamethasone. Plasma ACTH and cortisol levels did not increase after human CRH administration, but fasting plasma cortisol levels increased after ACTH treatment. The infusion of GIP increased plasma cortisol levels to 7.8 times above baseline. Radiological and cholesterol uptake studies pointed to a unilateral adrenal adenoma. Treatment with octreotide initially prevented the meal-induced increases in cortisol and GIP levels and decreased urinary cortisol excretion. Unilateral adrenalectomy was performed. Cortisol production by cultured adrenal adenoma cells from the patient was stimulated by GIP and ACTH. In situ hybridization studies using a GIP receptor probe showed an abundant expression of GIP receptor messenger ribonucleic acid in the adrenocortical adenoma. We conclude that food-dependent Cushing's syndrome results from the expression of GIP receptors on adrenocortical adenoma cells.

Peptide YY (PYY)-preferring receptors are expressed in the renal proximal tubule cell clone Cl.10 isolated from the PKSV-PCT cell line. They mediate PYY-inhibited cAMP production through coupling with pertussis-sensitive Gi proteins. Previous G alpha i RNA antisense experiments demonstrated the exclusive coupling of the PYY receptor to the Gi2 protein. Here we characterized a clone stably expressing G alpha i2 antisense RNA which exhibited only a partial decrease in G alpha i2 content (#60%) as estimated by Western blot. When compared to control Cl.10 cells, this clone, referred to as Cl.10(t), exhibited: (i) an increase in the dissociation constant of PYY receptors (6.42 vs 0.63 nM); (ii) a complete absence of inhibition of [125I]PYY binding by GTP gamma S and GTP; (iii) the failure of PYY to inhibit basal and forskolin-stimulated cAMP levels; (iv) the failure of PYY to stimulate [35S]GTP gamma S binding to membranes. These findings show that partial knockdown of G alpha i2 expression in Cl.10 cells completely abolish the coupling of PYY receptors to biological response.

Galanin has been shown to stimulate feeding when injected intracranially in rats. Lesion and Fos studies have shown that the neural pathway for feeding stimulated by mercaptoacetate (MA)-induced blockade of fatty acid oxidation includes several structures rich in galanin cell bodies or terminals. In the present experiment, we examined the role of hindbrain galanin in feeding stimulated by MA. We found that galanin (1 nmol) stimulates feeding when injected in the nucleus of the solitary tract (NTS), a site that is crucial for MA-induced feeding, or into the fourth ventricle (4V, 1 or 5 nmol) and that NTS or 4V injections of the galanin receptor antagonist, M40 (1.5 or 5 nmol), completely blocked feeding induced by MA (68 mg/kg). The effect of the M40 appeared to be specific for MA-induced feeding, since M40 did not significantly attenuate either feeding induced by the antimetabolic glucose analog, 2-deoxy-D-glucose (2DG, 100 or 200 mg/kg), or deprivation-induced water intake. Results suggest that feeding induced by decreased fatty acid oxidation relies upon galaninergic terminals in the hindbrain. Furthermore, results indicate that hindbrain neurons involved in MA-induced feeding differ neurochemically from those important for 2DG-induced feeding.

Distinct themes exist for ligand-binding domains of G protein-coupled receptors. The secretin receptor is prototypic of a recently described family in this superfamily which binds moderate-sized peptides possessing a diffuse pharmacophore. We recently demonstrated the importance of the N terminus and first loop of this receptor for secretin binding (Holtmann, M. H., Hadac, E. M., and Miller, L. J. (1995) J. Biol. Chem. 270:14394-14398). Here, we extend those findings to define another receptor domain important for agonist recognition and to focus on critical determinants within each of these domains. Extending the secretin-vasoactive intestinal polypeptide (VIP) chimeric receptor approach, we confirmed and refined the critical importance of the N terminus and the need to complement this with other domains of the secretin receptor. There was redundancy in the complementary determinants required, with the second extracellular loop able to compensate for the absence of the first loop. The first 10 residues of the N terminus of the secretin receptor were critical. Sequential segmental and site replacements permitted focusing on the His189-Lys190 sequence at the C terminus of the first extracellular loop, and on four residues (Phe257, Leu258, Asn260, and Thr261) in the N-terminal half of the second loop as providing critical determinants. All receptor constructs which expressed sensitive cAMP responses to secretin (EC50 <5 nM) bound this peptide with high affinity. Of note, one construct dissociated high affinity binding of secretin from its biological responsiveness, providing a clue to the conformational "switch" that activates this receptor.

Background: Human galanin (hGal) is a 30-residue non-amidated gut-brain peptide that shows considerable sequence divergence compared with galanin (Gal) forms of other species. Conflicting results have been reported with regard to the structural requirements for its modulatory action on gut motility., Methods: We investigated the effect of human and rat Gal and substituted analogues of Gal on the contractility of longitudinal muscle strips of the human colon in vitro., Results: Both hGal and rGal contracted the preparations in a concentration-dependent and tetrodotoxin-resistant manner without difference in sensitivity. The NH2-terminally truncated peptides hGal (3-30) and rGal (3-29) were inactive, whereas the NH2-terminal fragments, hGal (1-21) and rGal (1-18), remained fully responsive. Single amino acid substitutions at NH2-terminal positions showed divergent results: substitution of Trp2 reduced significantly potency and efficacy, whereas substitutions at positions 1, 3, 4, or 5 did not markedly modify the bioactivity of Gal. Galantide, a high-affinity Gal antagonist in the central nervous system, is a full agonist in human colonic smooth muscle., Conclusion: The COOH-terminal part of Gal contributes mainly the receptor-binding affinity of the peptide, whereas the NH2-terminal region is essential for biologic activity.

Gastric inhibitory polypeptide, originally isolated from porcine intestine, is a gastrointestinal hormone belonging to the vasoactive intestinal peptide (VIP)/glucagon/secretin family. GIP consists of 42 amino acid residues which is derived by proteolytic processing of a GIP precursor. In vivo and in vitro experiments have indicated that GIP auguments glucose-stimulated insulin secretion, suggesting that GIP plays an important role in the regulation of insulin secretion as an incretin. Thus, GIP now is generally referred to as glucose-dependent insulinotropic polypeptide. It is also suggested that GIP may be involved in the pathogenesis of non insulin-dependent diabetes mellitus (NIDDM). GIP exerts its biological actions by binding to its specific receptors, which appear to be coupled to G proteins. We have isolated a cDNA encoding a GIP receptor from a hamster insulinoma(HIT-T15) cDNA library. The hamster GIP receptor is a 462 amino acid protein having seven transmembrane segments. Expression of recombinant of hamster GIP receptors in Chinese hamster ovary (CHO) cells shows that it binds specifically to GIP with high affinity (IC50 = 9.6 nM) and is positively coupled to adenylate cyclase. RNA blot analysis reveals that a 3.8-kb GIP receptor mRNA is expressed at high levels in rat pancreatic islets as well as in HIT-T15 cells.

Motilin, a 22 amino acid polypeptide, is known to play an important role in the initiation of phase III activity of the interdigestive migrating contractions (IMC) in the dog and man. The precursor of human motilin consists of 115 amino acids including a 25 amino acid signal peptide in direct linkage with the 22 amino acid motilin sequence and a 66 amino acid carboxy-terminal motilin-associated peptide (MAP). Northern blot analysis revealed that motilin mRNA is abundant in the duodenum. In vitro studies suggested that motilin acts directly on motilin receptors located on gastrointestinal smooth muscle cells in the rabbit, cat and man. On the contrary, in vivo studies suggested that motilin receptors are likely to be present in the nervous system. The cloning of motilin receptors is needed to clarify the detailed mechanism(s) of motilin's action.

Pancreastatin is a 49 amino acid peptide first isolated, purified and characterized from porcine pancreas. Its biological activity in different tissues can be assigned to the C-terminal part of the molecule. Pancreastatin has a prohormonal precursor, chromogranin A, which is a glycoprotein present in neuroendocrine cells, including the endocrine pancreas. We have been interested in pancreastatin action in the liver. We found that pancreastatin has a glycogenolytic effect in the hepatocyte both in vivo and in vitro. We then studied and characterized the specific pancreastatin receptor in the rat liver plasma membrane, as well as the specific signal transduction. This receptor appears to be coupled to two different G proteins. A pertussis toxin-insensitive G proteins leads to the activation of phospholipase C, and therefore mediates the glycogenolytic effect in the liver by increasing cytoplasmic free calcium and stimulating protein kinase C. The role of cyclic GMP in the action of pancreastatin is not known yet, although it seems to regulate negatively the activation of phospholipase C. The precise mechanism by which pancreastatin stimulates guanylate cyclase activity remains to be studied.

Two molecular forms of peptide YY (PYY), PYY-(1--36) and PYY-(3--36), are abundant in rabbit intestine and blood. We have previously shown that PYY-(1--36) (PYYI) activates equipotently Y1 and Y2 receptors and PYY-(3--36) (PYY II) is a highly selective agonist for Y2 receptors. In the present study, we examined the effect of exogenous infusion of PYY on vagally stimulated gastric acid secretion in awake rabbits with chronic gastric fistula. To determine the specific PYY receptor(s) that mediates this effect, we used a highly selective Y1 agonist, Pro34-PYY, a synthetic PYY, and a Y2-selective agonist, PYY II. Vagal stimulation of acid secretion was elicited by an intravenous bolus injection of insulin (0.125 U/kg) 30 min after beginning a 180-min intravenous infusion of either PYY I, PYY II, or [Pro34]-PYY after a 50 micrograms/kg i.v. bolus of atropine followed immediately by a 500 micrograms/kg sc injection. During infusion of 200 pmol.kg 1.h-1 PYY I, acid output was significantly inhibited to 45 +/- 13% of maximum acid output 60 min after injection of insulin. Similarly, acid output during infusion of 200 pmol.kg-1.h-1 [Pro34]-PYY was significantly inhibited to 52 +/- 12% of maximum. In contrast, acid output during infusion of 200 pmol.kg-1.h-1 of PYY II was not significantly inhibited (101 +/- 18% of maximum). Infusion of double the dose (400 pmol.kg-1.h-1) of PYY II resulted in acid inhibition (51 = 15% of maximum), whereas infusion of the same dose did not significantly enhance acid inhibition by infusion of either PYY I or [Pro34]-PYY (28 +/- 11 and 42 +/- 15% of maximum). These results indicate that PYY, acting predominantly at Y1 receptors, is a potent inhibitor of vagally stimulated acid secretion in adult rabbits.

1. Galanin is a 29 (in humans 30) amino acids long neuropeptide with mostly inhibitory, hyperpolarizing actions. 2. Differential structural requirements of truncated forms of galanin and differential agonist/antagonist behaviour of chimeric peptides, high affinity galanin receptor ligands suggest the presence of pharmacologically distinct galanin receptor subtypes. 3. The galanin receptor from human Bowes melanoma cell line--a member of G-protein coupled receptor superfamily--has been cloned. 4. Galanin acts via Gi/G(o) proteins inhibiting cAMP production, inositol phosphate turnover, opening K+ channels or closing Ca2+ channels.

Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in the regulation of postprandial insulin secretion and proinsulin gene expression of pancreatic beta-cells. This study demonstrates the molecular cloning of a cDNA for the GIP-receptor from a human insulinoma lambda gt11 cDNA library. The cloned cDNA encoded a seven transmembrane domain protein of 466 amino acids which showed high homology (41%) to the human glucagon-like peptide 1 (GLP-1) receptor. Homology to the GIP receptor from rat or hamster was 79% and 81%, respectively. When transfected stably into fibroblast CHL-cells a high affinity receptor was expressed which coupled to the adenylate cyclase with normal basal cAMP and increasing intracellular cAMP levels under stimulation with human GIP-1-42 (EC50 = 1.29 x 10(-13) M). The receptor accepted only human GIP 1-42 (Kd = 1.93 +/- 0.2 x 10(-8) M) and porcine truncated GIP 1-30 (Kd = 1.13 +/- 0.1 x 10(-8) M) as high affinity ligands. At 1 microM, exendin-4 and (9-39)amide weakly reduced GIP-binding (25%) whereas secretin, glucagon, glucagon-like peptide-1, vasoactive intestinal polypeptide, peptide histidine-isoleucine, and pituitary adenylyl cyclase activating peptide were without effect. In transfected CHL cells, GIP-1-42 did not increase intracellular calcium. Northern analysis revealed one transcript of human GIP receptor mRNA with an apparent size of 5.5 kb. The exact understanding of GIP receptor regulation and signal transduction will aid in the understanding of the incretin hormone's failure to exert its biological action at the pancreatic B-cell in type II diabetes mellitus.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.

Incretins are endogenous peptides released from the gastrointestinal tract into the circulation during a meal that potentiate glucose-stimulated insulin secretion. At present, there are two established incretins: glucose-dependent insulinotropic polypeptide (GIP) and the truncated glucagon-like peptides (tGLPs), which are now being investigated for use in the treatment of diabetes mellitus. In the present study we cloned a rat islet GIP receptor complementary DNA (GIP-R1) to answer several important questions regarding the ligand-binding and intracellular signaling properties of the GP receptor. GIP-R1, when expressed transiently in monkey kidney (COS-7) or stably in Chinese hamster ovary (CHO-K1) cells, demonstrated comparable high affinity binding for either synthetic porcine (sp) GIP or synthetic human (sh) GIP. The IC50 values for displacement of [125I]spGIP in CHO-K1 cells were 2.6 +/- 0.8 and 3.1 +/- 0.9 nM for two different preparations of shGIP, and 3.7 +/- 1.5 and 3.6 +/- 0.4 nM for two preparations of spGIP. Saturation isotherms obtained with both intact cells and membranes gave monophasic binding curves with apparent Kd values of 204 +/- 17 and 334 +/- 94 pM, respectively. Cells expressed 12-15 x 10(3) receptors/cell. In COS-7 cells, spGIP and shGIP also exhibited similar IC50 values (7.6 +/- 1.2 and 8.9 +/- 1.8 nM, respectively). The receptor in CHO-K1 cells bound GIP-(1-30) with lower affinity (IC50 = 39 +/- 17 nM), whereas the fragments GIP-(19-30), GIP-(18-28), and GIP-(21-26) showed no apparent binding. The specificity of the receptor was further examined using several structurally related peptides. Surprisingly, exendin-(9-39) [Ex-(9-39)], a GLP-1 receptor antagonist, and Ex-4-(1-39), a GLP-1 receptor agonist, demonstrated some affinity for the GIP receptor, with 39% and 21% displacement of [125I]spGIP, respectively, at 1 microM. Other members of the secretin/vasoactive intestinal peptide family of peptides tested showed no interaction. GIP-R1 receptor binding correlated with activation of the adenylyl cyclase system, whereby spGIP and shGIP evoked concentration-dependent increases in cAMP accumulation with EC50 values of 8.7 +/- 1.5 x 10(-10)M and 8.1 +/- 1.6 x 10(-10)M for spGIP and shGIP, respectively. Increases in cAMP in the presence of 10 nM spGIP were not dependent on the ambient glucose concentration, with 22- and 18-fold increases in cAMP accumulation at 0.1 and 5.5 mM glucose, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and a single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).