Sustained orexigenic effect of Agouti related protein may be not mediated by the melanocortin 4 receptor

Intracerebroventricular (ICV) injection of Agouti related protein (AgRP), an endogenous melanocortin 3 and 4 receptor (MC3/4-R) antagonist, produces a prolonged increase in food intake. To clarify the roles of the MC3-R and MC4-R in AgRP-induced hyperphagia, the feeding effect of AgRP (83-132) was compared with that of the selective MC4-R antagonist, JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH11-22). Single ICV administration of AgRP (83-132) increased food intake for 48 h whilst ICV JKC-363 increased food intake for 8h. An increase in body weight at 24 and 48 h was observed following AgRP (83-132) but not JKC-363 treatment. These data suggest that the sustained orexigenic action of AgRP (83-132) may not be through MC4-R antagonism.     

Mitotic arrest caused by the amino terminus of Xenopus cyclin B2.

Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins.     

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.     

Rab32 regulates melanosome transport in Xenopus melanophores by protein kinase a recruitment

Intracellular transport is essential for cytoplasm organization, but mechanisms regulating transport are mostly unknown. In Xenopus melanophores, melanosome transport is regulated by cAMP-dependent protein kinase A (PKA). Melanosome aggregation is triggered by melatonin, whereas dispersion is induced by melanocyte-stimulating hormone (MSH). The action of hormones is mediated by cAMP: High cAMP in MSH-treated cells stimulates PKA, whereas low cAMP in melatonin-treated cells inhibits it. PKA activity is typically restricted to specific cell compartments by A-kinase anchoring proteins (AKAPs). Recently, Rab32 has been implicated in protein trafficking to melanosomes and shown to function as an AKAP on mitochondria. Here, we tested the hypothesis that Rab32 is involved in regulation of melanosome transport by PKA. We demonstrated that Rab32 is localized to the surface of melanosomes in a GTP-dependent manner and binds to the regulatory subunit RIIalpha of PKA. Both RIIalpha and Cbeta subunits of PKA are required for transport regulation and are recruited to melanosomes by Rab32. Overexpression of wild-type Rab32, but not mutants unable to bind PKA or melanosomes, inhibits melanosome aggregation by melatonin. Therefore, in melanophores, Rab32 is a melanosome-specific AKAP that is essential for regulation of melanosome transport.     

HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus

Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively. Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers. Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers. Ser90 phosphorylation was sufficient and necessary for causing this change in structure. Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal alpha-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg5-Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the alpha-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain.     

Cooperative interactions between replication origin-bound molecules of herpes simplex virus origin-binding protein are mediated via the amino terminus of the protein.

The virally encoded origin binding protein (OBP) of herpes simplex virus (HSV) is required for viral DNA synthesis. OBP binds at the replication origin to initimultienzyme replication complex (Challberg, M. D., and Kelly, T. J. (1989) Annu Rev. Biochem. 58, 671-717), OBP binds to two sites at the replication origin. The sequence-specific interaction of OBP with each binding site is localized to the major groove, and in both HSV origins the two interaction surfaces are in phase, aligned on the same face of the helix (Hazuda, D. J., Perry, H. C., Naylor, A. M., and McClements, W. L. (1991) J. Biol. Chem. 261, 24621-24625). Using native gel electrophoresis, we now demonstrate that OBP binding to the origin is highly cooperative and that cooperativity requires the putative NH2-terminal leucine zipper. Neither the phase nor orientation of the binding sites affect cooperativity, suggesting that the interaction promotes wrapping of origin DNA around the OBP multimer. A comparison of OBP DNase I footprints with the DNase I footprints of a truncated protein defective in cooperativity demonstrates that the interaction between OBPs bound at sites I and II affects the conformation of the intervening DNA, particularly when the phase or orientation of the two sites is different from wild type. OBP may elicit a unique nucleoprotein structure which facilitates unwinding of the origin and/or assembly of the replication complex. We also demonstrate that OBP can exchange binding sites, forming interduplex complexes. This property may be important for reinitiation of DNA replication.     

Identification of novel hexapeptide agonists at the Xenopus laevis melanophore melanocortin receptor

We used a combinatorial chemical approach to identify novel agonists for the endogenous melanocortin receptor expressed in Xenopus laevis melanophores. A random one-bead one-compound hexapeptide library was screened to detect new molecules able to induce pigment dispersion in melanophores. Our approach led to the discovery of seven related novel peptides able to stimulate pigment dispersion with EC50 in the range of 0.1-10 microM. Their action was inhibited by the amphibian melanocortin receptor antagonist dWRL. These novel peptides share no significant sequence homology with known melanocortins. This study may aid in the understanding of the chemical interaction between the melanocortin receptors and their ligands.     

Cleavage of the amino terminus of the prion protein by reactive oxygen species

Relatively limited information is available on the processing and function of the normal cellular prion protein, PrP(C). Here it is reported for the first time that PrP(C) undergoes a site-specific cleavage of the octapeptide repeat region of the amino terminus on exposure to reactive oxygen species. This cleavage was both copper- and pH-dependent and was retarded by the presence of other divalent metal ions. The oxidative state of the cell also decreased detection of full-length PrP(C) and increased detection of amino-terminally fragmented PrP(C) within cells. Such a post-translational modification has vast implications for PrP(C), in its processing, because such cleavage could alter further proteolysis, and in the formation of the scrapie isoform of the prion protein, PrP(Sc), because abnormal cleavage of PrP(Sc) occurs into the octapeptide repeat region.     

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. -Melanocyte stimulating hormone (-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111–113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7–9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3–5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH₂ peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.     

Contribution of melanocortin receptor exoloops to Agouti-related protein binding.

Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the alpha-melanocyte stimulating hormone analogue [Nle4,D-Phe7] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.     

Proliferation in vitro of melanophores from Xenopus laevis

Melanophores of wild-type and periodic albino mutants of Xenopus laevis were successfully cultured in vitro. They proliferated in the presence of alpha-melanocyte-stimulating hormone (alpha-MSH or cyclic adenosine monophosphate (cAMP) at a doubling time of 8-10 days. These proliferating melanophores retained their phenotypes, ability to synthesize melanin, and melanin-dispersing response to MSH stimulation. Neither depigmentation nor selective cell death of periodic albino melanophores was observed for at least 4 months during the cultivation.     

Statistics of active transport in Xenopus melanophores cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.     

Structure of the amino terminus of a gap junction protein

Charged amino acid residues in the amino terminus of gap junction forming proteins (connexins) form part, if not all, of the transjunctional voltage sensor of gap junction channels and play a fundamental role in ion permeation. Results from studies of the voltage dependence of N-terminal mutants predict that residues 1-10 of Group I connexins lie within the channel pore and that the N-terminus forms the channel vestibule by the creation of a turn initiated by the conserved G12 residue. Here we report that intercellular channels containing mutations of G12 in Cx32 to residues that are likely to interfere with flexibility of this locus (G12S, G12Y, and G12V) do not express junctional currents, whereas a connexin containing a proline residue at G12 (Cx32G12P), which is expected to maintain a structure similar to that of the G12 locus, forms nearly wild-type channels. We have solved the structure of an N-terminal peptide of Cx26 (MDWGTLQSILGGVNK) using 1H 2D NMR. The peptide contains two structured domains connected by a flexible hinge (domain-hinge-domain motif) that would allow the placement of the amino terminus within the channel pore. Residues 1-10 adopt a helical conformation and line the channel entrance while residues 12-15 form an open turn. Overall, there is good agreement between the structural and dynamic features of the N-terminal peptide provided by NMR and the functional studies of the voltage dependence of channels formed by wild-type and N-terminal mutations.     

Aging-related attenuation of EGF receptor signaling is mediated in part by increased protein tyrosine phosphatase activity

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343-19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase alpha (RPTPalpha). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.     

Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region

Protein-tyrosine phosphatases (PTPs) play a major role in regulating insulin signaling. Among the PTPs that regulate this signaling pathway, PTP1B plays an especially prominent role. PTP1B inhibits insulin signaling and has previously been shown to bind to the activated insulin receptor (IR), but neither the mechanism nor the physiological importance of such binding have been established. Here, we show that a previously undefined region in the N-terminal, catalytic half of PTP1B contributes to IR binding. Point mutations within this region of PTP1B disrupt IR binding but do not affect the catalytic activity of this phosphatase. This binding-defective mutant of PTP1B does not efficiently dephosphorylate the IR in cells, nor does it effectively inhibit IR signaling. These results suggest that PTP1B targets the IR through a novel binding element and that binding is required for the physiological effects of PTP1B on IR signal transduction.     

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.     

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY.

BACKGROUND: The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS: Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS: This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.     

Structures of the intradiskal loops and amino terminus of the G-protein receptor, rhodopsin.

The intradiskal surface of the transmembrane protein, rhodopsin, consists of the amino terminal domain and three loops connecting six of the seven transmembrane helices. This surface corresponds to the extracellular surface of other G-protein receptors. Peptides that represent each of the extramembraneous domains on this surface (three loops and the amino terminus) were synthesized. These peptides also included residues which, based on a hydrophobic plot, could be expected to be part of the transmembrane helix. The structure of each of these peptides in solution was then determined using two-dimensional 1H nuclear magnetic resonance. All peptide domains showed ordered structures in solution. The structures of each of the peptides from intradiskal loops of rhodopsin exhibited a turn in the central region of the peptide. The ends of the peptides show an unwinding of the transmembrane helices to form this turn. The amino terminal domain peptide exhibited alpha-helical regions with breaks and bends at proline residues. This region forms a compact domain. Together, the structures for the loop and amino terminus domains indicate that the intradiskal surface of rhodopsin is ordered. These data further suggest a structural motif for short loops in transmembrane proteins. The ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops are sufficient to code for the turn.