Endomorphin-1: induction of motor behavior and lack of receptor desensitization.

The endomorphins are recently discovered endogenous agonists for the mu-opioid receptor (Zadina et al., 1997). Endomorphins produce analgesia; however, their role in other brain functions has not been elucidated. We have investigated the behavioral effects of endomorphin-1 in the globus pallidus, a brain region that is rich in mu-opioid receptors and involved in motor control. Bilateral administration of endomorphin-1 in the globus pallidus of rats induced orofacial dyskinesia. This effect was dose-dependent and at the highest dose tested (18 pmol per side) was sustained during the 60 min of observation, indicating that endomorphin-1 does not induce rapid desensitization of this motor response. In agreement with a lack of desensitization of mu-opioid receptors, 3 hr of continuous exposure of the cloned mu receptor to endomorphin-1 did not diminish the subsequent ability of the agonist to inhibit adenylate cyclase activity in cells expressing the cloned mu-opioid receptor. Confirming the involvement of mu-opioid receptors, the behavioral effect of endomorphin-1 in the globus pallidus was blocked by the opioid antagonist naloxone and the mu-selective peptide antagonist Cys(2)-Tyr(3)-Orn(5)-Pen(7) amide (CTOP). Furthermore, the selective mu receptor agonist [d-Ala(2)-N-Me-Phe(4)-Glycol(5)]-enkephalin (DAMGO) also stimulated orofacial dyskinesia when infused into the globus pallidus, albeit transiently. Our findings suggest that endogenous mu agonists may play a role in hyperkinetic movement disorders by inducing sustained activation of pallidal opioid receptors.

Template-constrained cyclic peptide analogues of somatostatin: subtype-selective binding to somatostatin receptors and antiangiogenic activity.

Beta-turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interesting biological activities, contains a beta-turn in its bioactive conformation. The beta-turn and biological activities of somatostatin have been succesfully mimicked in cyclic hexapeptide analogues. Two novel, structured, non-peptidic molecules were developed that are capable of holding the bioactive tetrapeptide sequence of somatostatin analogues in a beta-turn conformation, as measured by somatostatin receptor (SSTR) binding. Template-constrained cyclic peptides in which the ends of the -Tyr-D-Trp-Lys-Val-tetrapeptide were linked by scaffolds based on either an N,N'-dimethyl-N,N'-diphenylurea or a substituted biphenyl system (DJS631 and DJS811, respectively), bound selectively to mouse SSTR2B and rat and human SSTR5 with affinities as high as 1 nM. DJS811, at a dose of 3 mg/kg/day, was shown in a mouse Matrigel model to inhibit angiogenesis to a level of 79%. The development of structured turn scaffolds allows beta-turn sequences to be contained in the context of a compact structure, with less peptidic nature and potentially greater bioavailability than cyclic hexapeptides. These systems can be used to study the determinants of beta-turn formation, as well as to probe the importance of turn sequences occurring in molecular recognition interactions. The antiangiogenic activity of DJS811 suggests that it may have antitumor activity as well. In addition, because SSTR2 is overexpressed on many types of tumors, DJS631 and DJS811 may be useful in the development of agents for tumor imaging or the radiotherapy of cancer.

Fentanyl and its analogs desensitize the cloned mu opioid receptor.

Fentanyl, and its structural analogs lofentanil and sufentanil, are potent analgesics used clinically in the management of pain. However, the high analgesic potency of these compounds is limited by the development of tolerance after chronic use. To investigate whether their tolerance development may be related to mu receptor desensitization, the cloned mouse mu receptor as well as mutant forms of the receptor were stably expressed in HEK 293 cells and tested for their response to continuous opioid treatment. Fentanyl and its analogs potently bound to the mu receptor and effectively inhibited cAMP accumulation. Three-hour pretreatment of mu receptors with fentanyl and its analogs desensitized the mu receptor by uncoupling it from adenylyl cyclase. The fentanyl analogs caused a slight internalization of the mu receptor as accessed by antibody binding to the epitope-tagged mu receptor. Truncation of the mu receptor by removal of its carboxyl terminus at Glu341 did not affect the ability of the fentanyl analogs to bind to and activate the mu receptor nor did it prevent the fentanyl analogs from desensitizing the receptor. In a previous study we showed that morphine did not desensitize the cloned mu receptor even though it is a potent and effective agonist at the mu receptor. Mutagenesis studies revealed that morphine interacts differently with the mu receptor to activate it than do the fentanyl analogs which may explain its lack of desensitization of the mu receptor. These results indicate that desensitization of the mu receptor may be a molecular basis for the development of tolerance to fentanyl and its analogs.

Differential regulation of the cloned kappa and mu opioid receptors.

To directly compare the regulation of the cloned kappa and mu opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K+ current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the kappa and the mu receptor to this somatostatin receptor. We show that the kappa receptor readily uncoupled from the K+ current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response. The desensitization of the kappa receptor was homologous, as the ability of somatostatin to mediate inhibition of adenylyl cyclase or potentiation of the K+ current was not altered by kappa receptor desensitization. The mu receptor uncoupled from the K+ current but not adenylyl cyclase after a 1 h pretreatment with agonist. Somatostatin was no longer able to potentiate the K+ current after mu receptor desensitization, thus this desensitization was heterologous. Interestingly, pretreatment with a somatostatin agonist caused uncoupling of the mu receptor but not the kappa receptor from the K+ current. These results show that in the same cell line, after a 1 h pretreatment with agonist, the kappa receptor displays homologous regulation, whereas the mu receptor undergoes only a heterologous form of desensitization. mu receptor desensitization may lead to the alterations of diverse downstream events, whereas kappa receptor regulation apparently occurs at the level of the receptor itself. Broad alterations of non-opioid systems by the mu receptor could be relevant to the addictive properties of mu agonists. Comparison of kappa and mu receptor regulation may help define the properties of the mu receptor which are important in the development of addiction, tolerance, and withdrawal to opioid drugs. These are the first studies to directly compare the coupling of the kappa and mu receptors to two different effectors in the same mammalian expression system.

Mutagenesis of the mouse delta opioid receptor converts (-)-buprenorphine from a partial agonist to an antagonist.

An aspartic acid at residue 95 (Asp95) in the delta receptor has previously been shown to be critical for the binding affinity of selective delta agonists. To gain a better understanding of the functional consequence of agonist action at the delta receptor, the Asp95 residue was mutated to an asparagine (D95N) and opioids were tested for binding and functional activation of the wild-type and mutant delta receptors. Selective agonists such as [D-Ser2,D-Leu5]enkephalin-Thr6 (DSLET) and [D-Ala2,D-Leu5]enkephalin (DADLE) had greatly reduced affinity for the D95N mutant receptor but still inhibited cAMP accumulation, which indicated that the mutant receptor was still functionally coupled to adenylyl cyclase. Antagonist binding was not affected by the Asp95 mutation. Similarly, the partial agonist buprenorphine bound with equally high affinity to the D95N mutant and the wild-type delta receptor, which indicated that Asp95 is not essential for the binding affinity of this opioid. Buprenorphine did not affect cAMP accumulation in HEK 293 cells expressing the D95N mutant, and it blocked the ability of DSLET and bremazocine to inhibit cAMP accumulation via the D95N mutant, which indicated that buprenorphine acts as an antagonist at the D95N mutant. These findings confirm the essential role of Asp95 in the activation of the delta receptor by agonists and reveal a molecular basis of the unique property of buprenorphine.

Mutagenesis of a single amino acid in the rat mu-opioid receptor discriminates ligand binding.

To investigate the role of Asp114 in the cloned rat mu-opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant mu receptor D114N. The wild-type mu receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The mu-selective agonists [D-Ala2,MePhe4,Gly-ol5]enkephalin and morphine and the endogenous peptides Met-enkephalin and beta-endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild-type mu receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild-type and D114N mutant mu receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N mu receptors to inhibit adenylyl cyclase. Although the Asp114 to Asn114 mutation reduced full-agonist binding, mutation of His297 to Asn297 in the mu receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the mu receptor than do the prototypical full agonists.

Opioid regulation of the mouse delta-opioid receptor expressed in human embryonic kidney 293 cells.

Opioid analgesics are used extensively in the management of pain. Although the clinically effective opioids bind with high affinity to the mu-opioid receptor, studies have suggested that the delta-opioid agonists might represent more ideal analgesic agents, with fewer side effects. A limitation to opiate effectiveness is the development of tolerance, an event that has been linked to opioid receptor desensitization. To gain a better understanding of delta-receptor agonist regulation, the cloned mouse delta receptor was stably expressed in human embryonic kidney 293 cells, and the functional effects of agonist pretreatment were examined. With a 3-hr pretreatment protocol, the delta-selective agonists [D-Pen2,D-Pen5]enkephalin, [D-Ala2,D-Leu5]enkephalin, and [D-Ser2,Leu5]enkephalin-Thr and the nonselective opioids levorphanol, etorphine, and ethylketocyclazocine were found to desensitize delta receptors. [D-Pen2,D-Pen5]enkephalin, [D-Ser2,Leu5]enkephalin-Thr, [D-Ala2,D-Leu5]enkephalin, and etorphine treatments also caused a pronounced internalization of the epitope-tagged delta receptor, suggesting that the desensitization and internalization may be related. In contrast, levorphanol pretreatment did not internalize the receptor but still resulted in a 400-fold reduction in potency, suggesting that prolonged treatment with levorphanol only uncoupled the delta receptor from adenylyl cyclase. In contrast to the desensitization induced by peptide-selective delta agonists, pretreatment with the delta-selective nonpeptide agonist 7-spiroindanyloxymorphone and morphine sensitized the opioid inhibition of forskolin-stimulated cAMP accumulation. This differential regulation of the delta receptor may be due to variations in the ability of agonists to bind to the receptor. This hypothesis was supported by the finding that a point mutation that converted Asp128 to Asn128 (D128N) diminished the ability of delta-selective agonists to inhibit cAMP accumulation while increasing the potency of morphine to reduce cAMP accumulation. In particular, a lack of desensitization of the delta receptor by morphine may contribute to our understanding of the molecular basis of development of morphine-induced tolerance and dependence.

Lanthionine-somatostatin analogs: synthesis, characterization, biological activity, and enzymatic stability studies.

A series of cyclic somatostatin analogs containing a lanthionine bridge have been subjected to studies of structure-activity relationships. A direct synthesis of the thioether bridged analog (1) of sandostatin (SMS 201,995) and several lanthionine hexa-, hepta-, and octapeptides was carried out by using the method of cyclization on an oxime resin (PCOR) followed by condensation reactions in solution. The structures of the target peptides were analyzed by liquid secondary ion mass spectrometry (LSIMS) and subjected to high-energy collision-induced dissociation (CID) studies after opening of the peptide ring by proteolytic cleavage. The biological activities of these compounds have been evaluated by assaying their inhibitory potencies for the release of growth hormone (GH) from primary cultures of rat anterior pituitary cells, as well as by their binding affinities to cloned somatostatin receptors (SSTR1-5). The structural modification of sandostatin by introducing a lanthionine bridge resulted in a significantly increased receptor binding selectivity. The lanthionine octapeptide with C-terminal Thr-ol (1) showed similar high affinity for rat SSTR5 compared to somatostatin[1-14] and sandostatin. However, it exhibits about 50 times weaker binding affinity for mSSTR2b than sandostatin. Similarly, the lanthionine octapeptide with the C-terminal Thr-NH2 residue (2) has higher affinity for rSSTR5 than for mSSTR2B. Both peptides (compounds 1 and 2) have much lower potencies for inhibition of growth hormone secretion than sandostatin. This is consistent with their affinities to SSTR2, the receptor which is believed to be linked to the inhibition of growth hormone release by somatostatin and its analogs. The metabolic stability of lanthionine-sandostatin and sandostatin have been studied in rat brain homogenates. Although both compounds have a high stability toward enzymatic degradation, the lanthionine analog has a 2.4 times longer half-life than sandostatin. The main metabolites of both compounds have been isolated and identified by using an in vivo technique (cerebral microdialysis) and mass spectrometry.

Changes in the association of G protein subunits with the cloned mouse delta opioid receptor on agonist stimulation.

G proteins couple delta opioid receptors to multiple cellular effector systems and are critical components of the delta opioid signal transduction cascade. To investigate the physical association of delta opioid receptors with G proteins, the cloned mouse delta opioid receptor was solubilized, and the G proteins associated with the receptor were identified through coimmunoprecipitation of the receptor/G protein complexes with antisera directed against different G(alpha) and G(beta) subunits. The delta receptor associates with G(i alpha1), G(i alpha3), G(o alpha), G(beta1) and G(beta2) subtypes. On agonist binding to the receptor, a greater proportion of the receptor is associated with G(i alpha) than with G(o alpha), G(i alpha1) dissociates from the receptor and G(i alpha2) associates with the receptor, whereas G(i alpha3) and the G(beta) subunits remain coupled to the delta receptor. These findings reveal dynamic changes in the G proteins associated with the receptor after agonist binding that may be linked to the activation of the delta receptor. In addition to pertussis toxin-sensitive G proteins, the delta receptor physically interacts with the pertussis toxin-insensitive G proteins G(q alpha) and G(z alpha). These interactions may be critical in linking delta receptors to phospholipase C. The diversity of G proteins associated with the delta opioid receptor may form the basis for the selective coupling of these receptors to multiple cellular effector systems.

Differential agonist regulation of the human kappa-opioid receptor.

Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a kappa-selective opioid, 3H-labeled (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro [4,5] dec-8-yl] benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [3H]69,593 binding, indicating that the human K receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a kappa-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a kappa-selective agonist, (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human kappa receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.

Molecular regulation of opioid receptors.

Opioid actions are initiated at membrane receptors which couple to cellular effectors through G protein-mediated pathways. In the central nervous system opioids reduce neuronal activity through the inhibition of voltage-dependent Ca2+ channels, the activation of K+ channels and the inhibition of adenylyl cyclase. A significant clinical limitation to opioid therapy is the development of tolerance, a biological event that has been linked to agonist effects at the receptor level. Molecular studies on the consequences of opioid receptor regulation will provide a better understanding of the cellular mechanisms involved in the agonist-mediated events in tolerance development.

Differential opioid agonist regulation of the mouse mu opioid receptor.

Mu opioid receptors mediate the analgesia induced by morphine. Prolonged use of morphine causes tolerance development and dependence. To investigate the molecular basis of tolerance and dependence, the cloned mouse mu opioid receptor with an amino-terminal epitope tag was stably expressed in human embryonic kidney (HEK) 293 cells, and the effects of prolonged opioid agonist treatment on receptor regulation were examined. In HEK 293 cells the expressed mu receptor showed high affinity, specific, saturable binding of radioligands and a pertussis toxin-sensitive inhibition of adenylyl cyclase. Pretreatment (1 h, 3 h, or overnight) of cells with 1 microM morphine or [D-Ala2MePhe4,Gly(ol)5]enkephalin (DAMGO) resulted in no apparent receptor desensitization, as assessed by opioid inhibition of forskolin-stimulated cAMP levels. In contrast, the morphine and DAMGO pretreatments (3 h) resulted in a 3-4-fold compensatory increase in forskolin-stimulated cAMP accumulation. The opioid agonists methadone and buprenorphine are used in the treatment of addiction because of a markedly lower abuse potential. Pretreatment of mu receptor-expressing HEK 293 cells with methadone or buprenorphine abolished the ability of opioids to inhibit adenylyl cyclase. No compensatory increase in forskolin-stimulated cAMP accumulation was found with methadone or buprenorphine; these opioids blocked the compensatory effects observed with morphine and DAMGO. Taken together, these results indicate that methadone and buprenorphine interact differently with the mouse mu receptor than either morphine or DAMGO. The ability of methadone and buprenorphine to desensitize the mu receptor and block the compensatory rise in forskolin-stimulated cAMP accumulation may be an underlying mechanism by which these agents are effective in the treatment of morphine addiction.

Differential internalization of somatostatin in COS-7 cells transfected with SST1 and SST2 receptor subtypes: a confocal microscopic study using novel fluorescent somatostatin derivatives.

A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF, [D-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of [D-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position alpha (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells transfected with complementary DNA encoding either sst1 or sst2A receptors both displayed specific, high affinity binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined to the cell surface in both cell types, as indicated by the fact that it was entirely removable by a hypertonic acid wash and assumed a pericellular distribution in the confocal microscope. At 37 C, the fate of specifically bound ligand varied markedly according to the type of receptor transfected. In cells encoding the sst1 receptor, approximately 20% of specifically bound ligand was recovered in the acid-resistant (i.e. intracellular) fraction. This fraction remained clustered at the periphery of the cell, suggesting that it was being sequestered either within or immediately beneath the plasma membrane. By contrast, in cells transfected with sst2A receptors, up to 75% of specifically bound ligand was recovered inside the cells, where it clustered into small endosome-like particles. These particles increased in size and moved toward the nucleus with time, suggestive of receptor-ligand complexes proceeding down the endocytic pathway. These results demonstrate that neuropeptides may be processed differently depending on the subtype of receptor expressed in their target cells and suggest that these different processing patterns may reflect different modes of sensitization/desensitization and recycling of the receptors, and thereby of transmembrane signaling.

Structure-function analysis of the cloned opiate receptors: peptide and small molecule interactions.

Opiate receptors mediate the physiological actions of opioid peptides and the clinical effects of the synthetic opioid agonists and antagonists. Site-directed mutagenesis studies have revealed regions of opiate receptors that are essential for ligand recognition, and this could aid the design of more selective opioid ligands.

Evidence that a novel somatostatin receptor couples to an inward rectifier potassium current in AtT-20 cells.

The recent cloning of five somatostatin receptors has made it possible to begin screening for selective ligands in order to begin characterization of these receptor subtypes expressed endogenously. We have recently reported the characterization of ligands selective for SSTR2 and SSTR5 [Raynor K. et al. (1993) Molec. Pharmac. 43, 838-844; 44, 385-392]. Both of these somatostatin receptor subtypes are endogenously expressed in the mouse pituitary cell line AtT-20 [O'Carroll A.-M. et al. (1992) Molec. Pharmac. 42, 939-946; Patel Y. C. et al. (1994) J. biol. Chem. 269, 1506-1509; Tallent M. et al. (1996) Neuroscience 71, 1073-1081]. Using these selective ligands, as well as other somatostatin analogs, we have characterized the somatostatin receptor which couples to the inward rectifier K+ current in AtT-20 cells. This receptor is sensitive to hexapeptide analogs of somatostatin, but insensitive to octapeptide analogs. This pharmacological profile is distinct from any of the cloned somatostatin receptors and therefore may represent a novel receptor. Somatostatin has been shown to potentiate an inward rectifying K+ channel in many different types of neuronal and non-neuronal cells. The activation of this current is thought to be an important mechanism by which somatostatin inhibits neuronal firing and decreases neurotransmitter and hormone release [Mihara S. et al. (1987) J. Physiol. 390, 335-355]. Therefore, the novel somatostatin receptor coupling to the inward rectifier in AtT-20 cells may be important in somatostatin's role in regulating neurotransmission and hormone release.

Molecular and functional properties of somatostain receptor subtypes.

Identification of the ligand binding domains of the somatostain (SRIF) receptors may facilitate the rational development of new SRIF ligands. To identify ligand-binding domains of sst1, and sst2, we tested a series of chimeras. Using site-directed mutagenesis, we found that to bind with high affinity to sst2, the sst2 agonists MK678 and SMS-201-995 require a four amino acid sequence (FDFV) at the border of the third extracellular loop and transmembrane 7. Transference of residue 294 in msst2 to sst1 conferred onto sst1 the ability to bind SMS-201-995 and other octapeptides. Cyclic peptides with a phenylalanine adjacent to the D-Trp appear to interact with Phe294 of sst2, whereas hexapeptides with a tyrosine adjacent to the D-Trp, such as MK 678 and BIM 23027, did not interact with the Phe294. We have recently identified a peptide that selectively binds to human (h)sst1, with 100-fold higher affinity than for the other cloned SRIF receptor subtypes. The second extracellular loop of sst1 is critical for this peptide to bind. This contrasts with the sites involved in binding of sst2 agonists and indicates that the two receptors have distinct ligand-binding domains. G proteins couple SRIF receptors to multiple cellular effector systems, including adenylyl cyclase and ionic conductance channels. A critical cellular action of SRIF is the inhibition of Ca2+ influx, which may be responsible for its blockade of hormone and neurotransmitter release. Various studies suggest that both sst2 and sst5 endogenously expressed in AtT-20 cells can couple to L-type Ca2+ channels; the coupling was pertussis toxin-sensitive. The coupling of sst2 to the Ca2+ channels was relatively resistant to desensitisation; 5 hours of pretreatment with MK 678 did not attenuate MK 678 inhibition of the Ca2+ current. In contrast, the sst5 receptors were desensitised by 1 hour of pretreatment with BIM 23052. Thus, the coupling of the two receptors to the Ca2+ channel could be differentially regulated. The SRIF receptor subtype coupling to the Ca2+ channel could also be distinguished by a unique antagonist, the peptide L362,855, which binds with high affinity to cloned sst5.

Identification of ligand binding determinants in the somatostatin receptor subtypes 1 and 2.

The somatostatin (SRIF) receptors (SSTRs) 1 and 2 bind SRIF and SRIF 28 with high affinity, although a number of synthetic hexapeptide and octapeptide analogs of SRIF bind selectively to SSTR2. Extracellular loop three and its adjoining trans-membrane-spanning regions contain elements essential for the binding of such analogs to murine SSTR2. In particular, a stretch of amino acids from residues 294-297 (FDFV) in murine SSTR2 in trans-membrane domain seven can determine affinity for the SSTR2-selective analogs. Within this region, Phe294 has previously been predicted to be essential for the binding of octapeptides (Kaupmann, K., Bruns, C., Raulf, F., Weber, H., Mattes, H., and Lubbert, H. (1995) EMBO J. 14, 727-735) based on the observation that SSTR1 can bind the octapeptide SMS-201-995 with reasonable affinity after a Ser-to-Phe conversion in the analogous region of this receptor (SSTR1S305F). We find that SSTR1S305F has low affinity for a number of SSTR2-selective hexapeptides, suggesting that these analogs have different binding requirements than SMS-201-995. A correlation is seen between the ability of SSTR1S305F to bind hexapeptide analogs and the presence of a phenylalanine, but not tyrosine, at position two in these small cyclic molecules. Thus, a single hydroxyl group in hexapeptides can play a critical role in determining receptor binding to these receptor mutants. We also find that the second extracellular loop of SSTR1 is important for the selectivity of certain SRIF agonists for binding to SSTR1. Taken together, our data indicate that there are multiple elements in the somatostatin receptors that can determine the binding affinity and selectivity of peptide analogs.

Somatostatin receptor subtypes SSTR2 and SSTR5 couple negatively to an L-type Ca2+ current in the pituitary cell line AtT-20.

The somatostatin receptor subtypes SSTR2 and SSTR5 mediate distinct endocrine and exocrine functions of somatostatin and may also be involved in mediating the neuromodulatory actions of somatostatin in the brain. To investigate whether these receptors couple to voltage-sensitive Ca2+ channels, SSTR2 and SSTR5 selective agonists were tested for their effects on AtT-20 cells using whole cell patch clamp techniques. The SSTR2 selective agonist MK 678 inhibited Ca2+ currents in AtT-20 cells. The effects of MK 678 were reversible and blocked by pertussis toxin pretreatment, suggesting that SSTR2 couples to the L-type Ca2+ channels via G proteins. Other SSTR2-selective agonists, including BIM 23027 and NC8-12, were able to inhibit the Ca2+ currents in these cells. The SSTR5 selective agonist BIM 23052 also inhibited the Ca2+ currents in these cells and this effect was reversible and blocked by pertussis toxin treatment. The ability of SSTR5 to mediate inhibition of the Ca2+ current was greatly attenuated by pretreatment with the SSTR5-selective agonist BIM 23052, whereas SSTR2-mediated inhibition of the Ca2+ current was not altered by pretreatment with the SSTR2-selective agonist MK 678. Thus, the SSTR2 and SSTR5 couplings to the Ca2+ current are differentially regulated. The peptide L362,855, which we previously have shown to have high affinity for the cloned SSTR5, had minimal effects on Ca2+ currents in AtT-20 cells at concentrations up to 100 nM and did not alter the ability of MK 678 to inhibit Ca2+ currents. However, it completely antagonized the effects of the SSTR5-selective agonist BIM 23052 on the Ca2+ currents. L362,855 is an antagonist/partial agonist at SSTR5 since it can reduce Ca2+ currents in these cells at concentrations above 100 nM. L362,855 is also an antagonist/partial agonist at the cloned rat SSTR5 expressed in CHO cells since it is able to block the inhibition of cAMP accumulation induced by somatostatin at concentrations below 100 nM but at higher concentrations can inhibit cAMP formation itself. Structural analysis of L362,855 reveals that only a single hydroxyl group at residue seven in the peptide is needed to convert the compound from an antagonist/partial agonist to a full agonist at SSTR5. These studies reveal that two different somatostatin receptor subtypes, SSTR2 and SSTR5, can mediate the inhibition of an L-type Ca2+ channel in AtT-20 cells by somatostatin. The receptor subtype responses can be distinguished by selective agonists and antagonists and are regulated differently by agonist pretreatment. The inhibition of Ca2+ influx into endocrine cells and neurons may be a major cellular mechanism by which somatostatin modulates hormone and neurotransmitter release. Our results reveal that at least two receptor subtypes can mediate this cellular response.

Development of a selective agonist at the somatostatin receptor subtype sstr1.

Somatostatin (SRIF) induces its biological actions by interacting with a family of five recently cloned receptors. SRIF receptor subtype, SSTR1, has high affinity for SRIF, but no ligand has been available that selectively binds to this receptor. Desamino acid(1,2,5) [DTryptophan8, N-p-isopropl-4-aminomethyl-l-phenylalanine9]SRIF(des-AA1,2,5 [DT rp8, IAmp9]SRIF inhibits the binding of [125ITyr11]SRIF to the cloned human SSTR1 with an affinity of 1.8+0.7nM, but does not bind to the other cloned SRIF receptors. des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF bound selectively, potently and saturably to SSTR1 with a Kd of 0.5 + 0.1 nM and a maximal binding density of 226 +/- 56 fmol/mg of protein. The binding of des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF to SSTR1 was potently inhibited by SRIF, [DTrp8]SRIF, des-AA1,2,5[DTrp8,IAmp9,DSer13]SRIF and SRIF 28 with K, values of 0.7+0.3, 0.2+0.2, 4.3+0.7 and 0.6+0.1 nM, respectively. SRIF analogs that selectively bind to SSTR2 and SSTR5 were impotent in displacing des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF from human SSTR1. des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF binding to SSTR1 expressed in COS-7 cells was reduced by GTPgS, and this effect was prevented by pertussis toxin treatment. In contrast, the binding of[125ITyr11]SRIF to SSTR1 was not affected by these treatments. These findings indicate that des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF may bind to SSTR1 in a defferent manner than SRIF. des-AA1,2,5[DTrp8,IAmp9]SRIF and its tyrosine analog are the first ligands that selectively bind to SSTR1 with high affinity and should be useful in localizing and determining the functional properties of this receptor.