RESUMEN
Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide. The majority of position 2 substitutions exhibited normal to enhanced GLP-2 receptor (GLP-2R) binding; in contrast, position 2 substitutions were less well tolerated in studies of receptor activation as only Gly, Ile, Pro, alpha-aminobutyric acid, D-Ala, or nor-Val substitutions exhibited enhanced GLP-2R activation. In contrast, alanine replacement at positions 5,6,17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding. Position 2 substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21 exhibited normal to enhanced GLP-2R binding but greater than 75% reduction in receptor activation. D-Ala(2), Pro(2) and Gly(2), Ala(16) exhibited significantly lower EC(50)s for receptor activation than the parent peptide (p < 0.01-0.001). Circular dichroism analysis indicated that the enhanced activity of these GLP-2 analogues was independent of the alpha-helical content of the peptide. These results indicate that single amino acid substitutions within GLP-2 can confer structural changes to the ligand-receptor interface, allowing the identification of residues important for GLP-2R binding and receptor activation.
Asunto(s)
Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Dicroismo Circular , Péptido 2 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Humanos , Datos de Secuencia Molecular , Ratas , Receptores de Glucagón/metabolismo , Receptores de Glucagón/fisiología , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and up-regulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition. However, the mechanism underlying these actions has remained unclear. Here we report the cloning and characterization of cDNAs encoding rat and human GLP-2 receptors (GLP-2R), a G protein-coupled receptor superfamily member expressed in the gut and closely related to the glucagon and GLP-1 receptors. The human GLP-2R gene maps to chromosome 17p13.3. Cells expressing the GLP-2R responded to GLP-2, but not GLP-1 or related peptides, with increased cAMP production (EC50 = 0.58 nM) and displayed saturable high-affinity radioligand binding (Kd = 0.57 nM), which could be displaced by synthetic rat GLP-2 (Ki = 0.06 nM). GLP-2 analogs that activated GLP-2R signal transduction in vitro displayed intestinotrophic activity in vivo. These results strongly suggest that GLP-2, like glucagon and GLP-1, exerts its actions through a distinct and specific novel receptor expressed in its principal target tissue, the gastrointestinal tract.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Péptidos/fisiología , Receptores de Glucagón/fisiología , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Células COS , Clonación Molecular , AMP Cíclico/metabolismo , Biblioteca de Genes , Péptido 1 Similar al Glucagón , Péptido 2 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Humanos , Mucosa Intestinal/metabolismo , Cinética , Datos de Secuencia Molecular , Especificidad de Órganos , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Ratas , Receptores de Glucagón/química , Receptores de Glucagón/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Relación Estructura-Actividad , TransfecciónRESUMEN
Glucagon-like peptide-2 (GLP-2) has recently been identified as a stimulator of intestinal epithelial growth, prompting the development of RIA and HPLC methodologies to study this peptide in more detail. A GLP-2-specific antiserum (UTTH-7) was developed that recognizes amino acids 25-30 of human and rat GLP-2-(1-33). UTTH-7 cross-reacts with N- and C-terminally modified forms of GLP-2, proglucagon, and the major proglucagon fragment. Analysis of rat ileal extracts demonstrated the presence of GLP-2-(1-33) as well as significant amounts of GLP-2-(3-33) (16 +/- 7% of total GLP-2). The level of total immunoreactive GLP-2 in plasma from fasted rats was 700 +/- 71 pg/ml, and this increased 3.6-fold (P < 0.001) in 24-h fed rats. HPLC analysis demonstrated the presence of both GLP-2-(1-33) and GLP-2-(3-33) in plasma from fasted rats, with increments in both peptides in plasma from fed rats. Immunoreactive GLP-2 increased in plasma from human subjects 2 h after a meal, rising from 851 +/- 230 to 1106 +/- 211 pg/ml (P < 0.05); 15 +/- 4% of this immunoreactivity was accounted for by the presence of intact GLP-2. HPLC showed the presence of both GLP-2-(1-33) and GLP-2-(3-33) in plasma from fed humans. Incubation of human GLP-2-(1-33) with the enzyme dipeptidylpeptidase IV resulted in liberation of GLP-2-(3-33), whereas replacement of Ala2 with Gly2 prevented this cleavage. Thus, while GLP-2-(1-33) is a major circulating and tissue form of GLP-2, GLP-2-(3-33) is a significant component ofimmunoreactive GLP-2 in both intestine and plasma.
Asunto(s)
Péptidos/sangre , Péptidos/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión/métodos , Dipeptidil Peptidasa 4/farmacología , Ingestión de Alimentos , Ayuno , Femenino , Péptido 2 Similar al Glucagón , Péptidos Similares al Glucagón , Humanos , Masculino , Datos de Secuencia Molecular , Péptidos/genética , Radioinmunoensayo/métodos , RatasRESUMEN
Species-specific differences in the enzymatic inactivation of peptides is an important consideration in the evaluation of therapeutic efficacy. We demonstrate that glucagon-like peptide 2 (GLP-2), shown to be highly intestinotrophic in mice, promotes an increase in intestinal villus height but has no trophic effect on small bowel weight in rats. The reduced intestinotrophic activity of GLP-2 in rats is attributable to inactivation by the enzyme dipeptidyl peptidase IV (DPP-IV). GLP-2(1-33) was degraded to GLP-2(3-33) following incubation with human placental DPP-IV or rat serum but not by serum from DPP-IV-deficient rats. Administration of rat GLP-2 to DPP-IV-deficient rats was associated with markedly increased bioactivity of rat GLP-2 resulting in a significant increase in small bowel weight. A synthetic GLP-2 analog, r[Gly2]GLP-2, with an alanine to glycine substitution at position 2, was resistant to cleavage by both DPP-IV and rat serum in vitro. Treatment of wild-type rats with r[Gly2]GLP-2 produced a statistically significant increase in small bowel mass. DPP-IV-mediated inactivation of GLP-2 is a critical determinant of the growth factor-like properties of GLP-2.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Glucagón/farmacología , Péptidos/farmacología , Animales , Biotecnología , Glucagón/antagonistas & inhibidores , Glucagón/metabolismo , Péptido 2 Similar al Glucagón , Péptidos Similares al Glucagón , Humanos , Técnicas In Vitro , Intestino Delgado/efectos de los fármacos , Intestino Delgado/crecimiento & desarrollo , Ratones , Péptidos/antagonistas & inhibidores , Péptidos/metabolismo , Ingeniería de Proteínas , Ratas , Ratas Endogámicas F344 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C(PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca2+ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling between Ca(2+)-CaM signalling and PKC-mediated phosphorylation cascades. We have studies Ca2+ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD) spectroscopy. The wild-type MARCKS and MRP peptides induced significant increases in the Ca2+ affinity of CaM (pCa 6.1 and 5.8, respectively, compared to 5.2, for CaM in the absence of bound peptides), whereas a modified MARCKS peptide, in which the four serine residues susceptible to phosphorylation in the wild-type sequence have been replaced with aspartate residues to mimic phosphorylation, had smaller effect (pCa 5.6). These results are consistent with the notions that phosphorylation of MARCKS reduces its binding affinity for CaM and that the CaM binding affinity of the peptides is coupled to the Ca2+ affinity of CaM. All three MARCKS/MRP peptides perturbed the backbone NMR resonances of residues in both the N- and C-terminal domains of CaM and, in addition, the wild-type MARCKS and the MRP peptides induced strong positive cooperativity in Ca2+ binding by CaM, suggesting that the peptides interact with the amino- and carboxy-terminal domains of CaM simultaneously. NMR analysis of the Ca(2+)-CaM-MRP peptide complex, as well as CD measurements of Ca(2+)-CaM in the presence and absence of MARCKS/MRP peptides suggest that the peptide bound to CaM is non-helical, in contrast to the alpha-helical conformation found in the CaM binding regions of myosin light-chain kinase and CaM-dependent protein kinase II. The adaptation of the CaM molecule for binding the peptide requires disruption of its central helical linker between residues Lys-75 and Glu-82.
Asunto(s)
Calcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Conformación Proteica , Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dicroismo Circular , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
We describe the isolation and interaction with calmodulin (CaM) of two 10-amino-acid peptides (termed peptides 1 and 2; AWDTVRISFG and AWPSLQAIRG respectively) derived from a phage random peptide display library. Both peptides are shorter than previously described CaM-binding peptides and lack certain features found in the sequences of CaM-binding domains present in CaM-activated enzymes. However, 1H NMR spectroscopy and fluorimetry indicate that both peptides interact with CaM in the presence of Ca2+. The two peptides differentially inhibited CaM-dependent kinases I and II (CaM kinases I and II) but did not affect CaM-dependent phosphodiesterase. Peptide 1 inhibited CaM kinase I but not CaM kinase II, whereas peptide 2 inhibited CaM kinase II, but only partially inhibited CaM kinase I at a more than 10-fold higher concentration. Peptide 1 also inhibited a plant calcium-dependent protein kinase, whereas peptide 2 did not. The ability of peptides 1 and 2 to differentially inhibit CaM-dependent kinases and CaM-dependent phosphodiesterase suggests that they may bind to distinct regions of CaM that are specifically responsible for activation of different CaM-dependent enzymes.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/química , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calmodulina/metabolismo , Bovinos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Espectroscopía de Resonancia Magnética , Masculino , Neurospora , Mapeo Peptídico , Hidrolasas Diéster Fosfóricas/metabolismo , Espectrometría de Fluorescencia , Testículo/químicaRESUMEN
Calmodulin (CaM) acts as an intracellular calcium sensor that translates the Ca2+ signal into a variety of cellular processes. Ca(2+)-CaM recognition of a short polypeptide segment in target proteins induces conformational changes in both CaM and the target, enabling the target protein to become functionally active. The solution and crystal structures of Ca(2+)-CaM bound to peptides derived from three CaM-dependent enzymes reveal structural features that are common in target recognition by Ca(2+)-CaM. Phosphorylation of the target proteins at sites in or near the CaM-binding region modulates binding of CaM, thereby providing an additional mechanism of functional regulation. The structural aspects of target recognition by Ca(2+)-CaM are discussed using mainly the three-dimensional structural information obtained with nuclear magnetic resonance spectroscopy and X-ray diffraction methods.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Calmodulina/biosíntesis , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
A chromatographic assay has been developed to quantitate racemization occurring during attachment of protected amino acids to peptide synthesis resins. Acidolytic cleavage of deprotected amino acids from supports and subsequent derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) gave diastereomers separable by reverse-phase HPLC using aqueous acetonitrile. The assay is reliable to 0.1% racemate with a detection limit of approximately 100 pmol. The technique was applied to several acid-labile resins and was used to evaluate the racemizing characteristics of various anchoring methods.
Asunto(s)
Alanina/análogos & derivados , Aminoácidos/química , Dinitrobencenos , Indicadores y Reactivos , Péptidos/síntesis química , Cromatografía Líquida de Alta Presión , EstereoisomerismoRESUMEN
In vitro multicell spheroids from a human melanoma cell line and the human colon cancer cell line HT29, used as control, have been established as a model of poorly vascularized micrometastases in vivo. The antimelanoma monoclonal antibody 96.5 was radiolabeled with 131I at specific radioactivities from 1.85 to 3.96 GBq/mg. Cytotoxicity of 131I-96.5 to the spheroids, at an initial size of 300 microns in diameter, was investigated as a function of concentration of 131I-96.5 in the incubation medium, specific radioactivity, and treatment time. Spheroid growth delay and clonogenic survival of cells disaggregated from the spheroids at various times after treatment were used as end points. Therapeutic effects increased with the concentration of 131I-96.5 within the range 0.2 to 2 mg/liter (0.34 to 3.4 GBq/liter) at a fixed specific radioactivity. The effects increased with specific radioactivity at a fixed concentration of 131I-96.5. Difference in therapeutic effect was also observed between treatment times of 8 and 24 h. Radiation doses to the melanoma spheroids varied from 10 to 16 Gy. Unlabeled 96.5 at 2 mg/liter or 131I-iodide at 1.7 GBq/liter did not affect the growth of the melanoma spheroids. The HT29 spheroids, however, only suffered slight cytotoxicity at 1 or 2 mg/liter of 131I-96.5 and for a treatment time of 24 h despite comparable radiosensitivity of HT29 cells and melanoma cells to high-dose-rate radiation. Similar cytotoxicity was observed in the HT29 group treated with 131I-iodide at 1.7 GBq/liter. Present findings therefore demonstrate preferential and adequate killing of the melanoma spheroids by 131I-96.5 at 0.5 mg/liter and 3.96 GBq/mg in 8 h.