The first nonsulfated sulfakinin activity reported suggests nsDSK acts in gut biology

Invertebrate sulfakinins are structurally and functionally homologous to vertebrate cholecystokinin (CCK) and gastrin. To date, sulfakinins are reported to require a sulfated tyrosine for activity; sulfated and nonsulfated CCK and gastrin are active. This is the first nonsulfated sulfakinin activity reported. Nonsulfated Drosophila melanogaster sulfakinins or drosulfakinins (nsDSK I; PheAspAspTyrGlyHisMetArgPheNH2) and (nsDSK II; GlyGlyAspAspGlnPheAspAspTyrGlyHisMetArgPheNH2) decreased the frequency of contractions of adult D. melanogaster foregut (crop) in vivo. The EC50's for nsDSK I and nsDSK II were approximately 2 x 10(-9)M and approximately 3 x 10(-8)M, respectively. Nonsulfated DSK peptides also decreased the frequency of larval anterior midgut contractions. Sulfated DSK peptides decreased both adult and larval gut contractions. Whether sulfation is required for sulfakinin activity may depend on where the peptide is applied, what tissue is analyzed, or what preparation is used. D. melanogaster contains two sulfakinin receptors, DSK-R1 and DSK-R2; vertebrates contain two CCK receptors, CCK-1 and CCK-2. A sulfated DSK I analog, [Leu7] sDSK I, binds to expressed DSK-R1; the corresponding nonsulfated analog does not bind to DSK-R1. No DSK-R2 binding data are reported. Sulfated and nonsulfated CCK peptides preferentially bind to CCK-1 or CCK-2, respectively. Sulfated and nonsulfated sulfakinins may bind to DSK-R1 or DSK-R2, respectively. Sulfakinin activities, spatial and temporal distribution, and homology to CCK and gastrin suggest sulfated and nonsulfated DSK peptides act in diverse roles in the neural and gastrointestinal systems including gut emptying and satiety.     

The different effects of structurally related sulfakinins on Drosophila melanogaster odor preference and locomotion suggest involvement of distinct mechanisms

Sulfakinins are myoactive peptides and antifeedant factors. Naturally occurring drosulfakinin I (DSK I; FDDYGHMRFNH₂) and drosulfakinin II (DSK II; GGDDQFDDYGHMRFNH₂) contain sulfated or nonsulfated tyrosine. We discovered sDSK II and nsDSK II influenced Drosophila melanogaster larval odor preference. However, sDSK I, nsDSK I, MRFNH₂, and saline did not influence odor preference. We discovered sDSK I and nsDSK I influenced larval locomotion. However, sDSK II, nsDSK II, MRFNH₂, and saline did not influence locomotion. Our novel data suggest distinct mechanisms underlie the effects of DSK I and DSK II peptides on odor preference and locomotion, parameters important to many facets of animal survival.     

Drosulfakinin activates CCKLR-17D1 and promotes larval locomotion and escape response in Drosophila

Neuropeptides are ubiquitous in both mammals and invertebrates and play essential roles in regulation and modulation of many developmental and physiological processes through activation of G-protein-coupled-receptors (GPCRs). However, the mechanisms by which many of the neuropeptides regulate specific neural function and behaviors remain undefined. Here we investigate the functions of Drosulfakinin (DSK), the Drosophila homolog of vertebrate neuropeptide cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system. We provide biochemical evidence that sulfated DSK-1 and DSK-2 activate the CCKLR-17D1 receptor in a cell culture assay. We further examine the role of DSK and CCKLR-17D1 in the regulation of larval locomotion, both in a semi-intact larval preparation and in intact larvae under intense light exposure. Our results suggest that DSK/CCKLR-17D1 signaling promote larval body wall muscle contraction and is necessary for mediating locomotor behavior in stress-induced escape response.     

Drosulfakinin activates CCKLR-17D1 and promotes larval locomotion and escape response in Drosophila

Neuropeptides are ubiquitous in both mammals and invertebrates and play essential roles in regulation and modulation of many developmental and physiological processes through activation of G-protein-coupled-receptors (GPCRs). However, the mechanisms by which many of the neuropeptides regulate specific neural function and behaviors remain undefined. Here we investigate the functions of Drosulfakinin (DSK), the Drosophila homolog of vertebrate neuropeptide cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system. We provide biochemical evidence that sulfated DSK-1 and DSK-2 activate the CCKLR-17D1 receptor in a cell culture assay. We further examine the role of DSK and CCKLR-17D1 in the regulation of larval locomotion, both in a semi-intact larval preparation and in intact larvae under intense light exposure. Our results suggest that DSK/CCKLR-17D1 signaling promote larval body wall muscle contraction and is necessary for mediating locomotor behavior in stress-induced escape response.     

Cloning and functional expression of the first Drosophila melanogaster sulfakinin receptor DSK-R1

Described in this report is a successful cloning and characterization of a functionally active Drosophila sulfakinin receptor designated DSK-R1. When expressed in mammalian cells, DSK-R1 was activated by a sulfated, Met(7-->Leu(7)-substituted analog of drosulfakinin-1, FDDY(SO(3)H)GHLRF-NH(2) ([Leu(7)]-DSK-1S). The interaction of [Leu(7)]-DSK-1S with DSK-R1 led to a dose-dependent intracellular calcium increase with an EC(50) in the low nanomolar range. The observed Ca(2+) signal predominantly resulted from activation of pertussis toxin (PTX)-insensitive signaling pathways pointing most likely to G(q/11) involvement in coupling to the activated receptor. The unsulfated [Leu(7)]-DSK-1 was ca. 3000-fold less potent than its sulfated counterpart which stresses the importance of the sulfate moiety for the biological activity of drosulfakinin. The DSK-R1 was specific for the insect sulfakinin since two related vertebrate sulfated peptides, human CCK-8 and gastrin-II, were found inactive when tested at concentrations up to 10(-5) M. To our knowledge, the cloned DSK-R1 receptor is the first functionally active Drosophila sulfakinin receptor reported to date.     

Structure-activity and immunochemical data provide evidence of developmental- and tissue-specific myosuppressin signaling

Myosuppressin peptides dramatically diminish contractions of the gut and heart. Thus, delineating mechanisms involved in myosuppressin signaling may provide insight into peptidergic control of muscle contractility. Drosophila myosuppressin (DMS, TDVDHVFLRFamide) structure-activity relationship (SAR) was investigated to identify an antagonist and explore signaling. Alanyl-substituted, N-terminal truncated, and modified amino acid analogs identified residues and peptide length required for activity. Immunochemistry independently provided insight into myosuppressin mechanisms. DMS decreased gut motility and cardiac contractility dose dependently; the different effective concentrations at half maximal-response were indicative of tissue-specific mechanisms. Replacement of aspartic acid 2 (D2) generated an analog with different developmental- and tissue-specific effects; [A2] DMS mimicked DMS in adult gut (100% inhibition), yet decreased larval gut contractions by only 32% with increased potency in pupal heart (126% inhibition). The DMS active core differed across development and in tissues; adult (DHVFLRFamide) and larval gut (TDVDHVFLRFamide), and adult (VFLRFamide) and pupal heart (VFLRFamide). Substitution of D2 and D4 with a modified amino acid, p-benzoyl-phenylalanine, produced developmental- and tissue-specific antagonists. In the presence of protease inhibitors, DMS and VFLRFamide were more effective in adult gut, but lower or unchanged in pupal heart compared to peptide or analog alone, respectively. DMS-specific antisera stained neurons that innervated the gut or heart. This study describes novel antagonists and data to identify developmental- and tissue-specific mechanisms underlying the pleotropic effects of myosuppressin in muscle physiology.     

Insect satiety: sulfakinin localization and the effect of drosulfakinin on protein and carbohydrate ingestion in the blow fly, Phormia regina (Diptera: Calliphoridae)

Sulfakinins, which are satiety factors in invertebrates, have previously been shown to inhibit feeding in the German cockroach and desert locust. This study examines the occurrence of sulfakinin immunoreactivity and the role of sulfakinin as a feeding satiety factor in the black blow fly, Phormia regina. Specifically, this study examines the effect of sulfakinin on two of the blow fly's nutrient requirements (i.e., carbohydrates and proteins). We observed sulfakinin immunoreactive cells in the brains of both male and female flies. We found that drosulfakinin I (DrmSKI, FDDY[SO(3)H]GHMRFa) significantly inhibited carbohydrate feeding by 44% at the most effective dose (10 nmol) in female flies. Statistically, there was no significant effect on males; however, injections of 10 nmol DrmSKI reduced carbohydrate feeding by 34% compared to the sham. Drosulfakinin had no effect on protein feeding and no significant inhibition was detected in females or males. The results of this study lend further support to the idea that carbohydrate and protein feeding are regulated by separate control mechanisms, especially in Calliphoridae.     

Interaction of vasoactive intestinal peptide (VIP) and N-terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes

Six vasoactive intestinal peptide (VIP) analogs inhibited [125I]iodo-VIP and [125I]iodo-helodermin binding to high-affinity VIP receptors in rat hepatic membranes. They also stimulated adenylate cyclase activity through these receptors, their decreasing order of potency being VIP greater than [D-Ala4]VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP greater than [D-Phe2]VIP greater than [D-Arg2]VIP, with the latter two peptides acting as partial agonists only. All VIP analogs tested on rat pancreatic membranes were able to stimulate adenylate cyclase, their order of potency being very similar to that observed on hepatic membranes. [D-Ser2]VIP, [D-His1]VIP, [D-Arg2]VIP and [D-Phe2]VIP were partial agonists with an intrinsic activity of, respectively, 0.8, 0.7, 0.35 and 0.09 as compared to that of VIP = 1.0. [D-Phe2]VIP competitively and selectively inhibited VIP-stimulated adenylate cyclase activity (Ki = 0.1 microM). On male rat anterior pituitary homogenates the order of potency of the peptides was VIP greater than [D-Ala4]VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP. [D-Ser2]VIP and [D-His1]VIP acted as partial agonists. Besides, [D-Phe2]VIP and [D-Arg2]VIP were inactive as well as unable to inhibit VIP-stimulated adenylate cyclase activity. These results indicated that (a) the efficacy of VIP receptor/effector coupling depended on the tissue tested; (b) the possibility exists to design a VIP antagonist by appropriate modification in the N-terminal moiety of the molecule.     

Spatial and temporal immunocytochemical analysis of drosulfakinin (Dsk) gene products in the Drosophila melanogaster central nervous system

The spatial and temporal distribution of three peptides, DSK I, DSK II, and DSK 0, encoded by the Drosophila melanogaster drosulfakinin (Dsk) gene, have been examined in the central nervous system. DSK I and DSK II have a -RFamide C-terminus and are structurally similar to sulfakinin peptides; in contrast, DSK 0 contains -SFamide and is not structurally similar to sulfakinins. Antisera specificities were determined by the design of the antigens and confirmed by dot blot analysis and preincubation with peptides prior to their use in immunocytochemistry. The distribution of immunoreactivity suggests that all three DSK peptides are processed from the polypeptide precursor and expressed in many of the same cells. Expression was observed at all developmental stages with an increase in the level of staining and the number of immunoreactive cells as development progresses. Cells in the brain lobe, optic lobe, subesophageal ganglion, thoracic ganglia, and the eighth abdominal neuromere contain DSK-immunoreactive materials. Immunoreactive fibers project from some cells and extend into the brain and ventral ganglion with regions of extensive arborization. DSK 0 immunoreactivity provides initial evidence for the presence of a -SFamide peptide in neural tissue. The observed expression of DSK-immunoreactive materials throughout development in numerous cells of the central nervous system suggests that DSK peptides may serve as hormones, modulators, or transmitters involved in several functions.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.     

Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N-terminus.

Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N-terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9-fluorenylmethyloxycarbonyl (Fmoc) and 9-fluorenylmethyl ester (OFm) at the alpha-amino (Nalpha-Fmoc-Ang II) or the beta-carboxyl ([Asp(OFm)1]-Ang II) groups of the Asp1 residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT1 Ang II receptor, Nalpha-Fmoc-Ang II bound with high affinity, whereas [Asp(OFm)1]-Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea-pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in Nalpha-Fmoc-Ang II. Thus, analogs with Fmoc- or OFm-type groups coupled to the Asp1 residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N-terminal portion and the AT1 receptor is proposed to explain the tachyphylactic phenomenon.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

Adenosine receptor ligands with oxygenated N6-substituents

A number of novel adenosine analogs bearing oxygenated substituents in the N6-position have been prepared and evaluated as A1 adenosine agonists. Improved conditions for the synthesis of N6-substituted adenosines and a new one pot procedure for the synthesis of 2-amino-7-oxabicyclo[2.2.1]hept-5-ene are also reported.     

Orn5]URP acts as a pure antagonist of urotensinergic receptors in rat cortical astrocytes

Cultured rat astrocytes, which express functional urotensin II (UII)/UII-related peptide (URP) receptors (UT), represent a very suitable model to investigate the pharmacological profile of UII and URP analogs towards native UT. We have recently designed three URP analogs [D-Trp4]URP, [Orn5]URP and [D-Tyr6]URP, that act as UT antagonists in the rat aortic ring bioassay. However, it has been previously reported that UII/URP analogs capable of inhibiting the contractile activity of UII possess agonistic activity on UT-transfected cells. In the present study, we have compared the ability of URP analogs to compete for [125 I]URP binding and to modulate cytosolic calcium concentration ([Ca2+]c) in cultured rat astrocytes. All three analogs displaced radioligand binding: [D-Trp4]URP and [D-Tyr6]URP interacted with high- and low-affinity sites whereas [Orn5]URP only bound high-affinity sites. [D-Trp4]URP and [D-Tyr6]URP both induced a robust increase in [Ca2+]c in astrocytes while [Orn5]URP was totally devoid of activity. [Orn5]URP provoked a concentration-dependent inhibition of URP- and UII-evoked [Ca2+]c increase and a rightward shift of the URP and UII dose-response curves. The present data indicate that [D-Trp4]URP and [D-Tyr6]URP, which act as UII antagonists in the rat aortic ring assay, behave as agonists in the [Ca2+]c mobilization assay in cultured astrocytes, whereas [Orn5]URP is a pure selective antagonist in both rat aortic ring contraction and astrocyte [Ca2+]c mobilization assays.     

Development of spiroguanidine-derived α7 neuronal nicotinic receptor partial agonists

We describe the synthesis of quinuclidine-containing spiroguanidines and their utility as α7 neuronal nicotinic acetylcholine receptor (nAChR) partial agonists. The convergent synthetic route developed for this study allowed for rapid SAR investigation and provided access to a structurally diverse set of analogs. A potent and selective α7 nAChR partial agonist, N‐(6‐methyl‐1,3‐benzoxazol‐2‐yl)‐3′,5′‐dihydro‐4‐azaspiro[bicyclo[2.2.2]octane‐2,4′‐imidazole]‐2′‐amine (BMS-910731, 16), was identified. This compound induced immediate early genes c-fos and Arc in a preclinical rodent model of α7 nAChR-derived cellular activation and plasticity. Importantly, the ability to incorporate selectivity for the α7 nACh receptor over the 5-HT_3A receptor in this series suggested a significant difference in steric requirements between the two receptors.     

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.     

Effect of cyclic nucleotide analogs on intrachain site I of protein kinase isozymes

The effects of numerous cAMP analogs present in the [3H]cAMP binding reaction on subsequent dissociation of [3H]cAMP from the regulatory subunit of cAMP-dependent protein kinase I and II were analyzed. Certain analogs with modification at either C-8 or C-2 showed relative selectivity for one (site 1) of two intrachain cAMP binding sites of both isozymes. Modification at C-6 caused selectivity for the second site (site 2). The combination of a site-1-directed and site-2-directed analog inhibited [3H]cAMP binding much more than did either analog alone. In general, there was a correlation between the site 1 selectivity and the ability of the analog to stimulate the binding of [3H]cIMP, which selects site 2. The site-1-directed analogs stimulated the initial rate of [3H]cIMP binding. The stimulatory effect was enhanced in the presence of a polycationic protein such as histone and was inhibited by high ionic strength. The type I and II isozymes exhibited large differences in analog specificity for this effect. For type I, of the analogs tested the most efficacious for stimulating [3H]cIMP binding were those containing a nitrogen atom attached to C-8, 8-aminobutylamino-cAMP being the most effective. Type II responded best to analogs containing a sulfur atom attached to C-8, 8-SH-cAMP being the most effective of those tested. The stimulatory effect was accentuated in the presence of MgATP when using type I, but this nucleotide had no effect when using type II. It is proposed that in intact tissues cAMP binding to site 1 of either isozyme stimulates the binding to site 2.