Galanin: Structural requirements for binding and signal transduction in RINm5F insulinoma cells

Gallwitz, Baptist; Schmidt, Wolfgang E.; Schwarzhoff, Rainer; Creutzfeldt, W.

doi:10.1016/s0006-291x(05)80204-9

Cited by 25 publications

(11 citation statements)

References 28 publications

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“…Interestingly, nonamidated and amidated hGals showed no differences in biological activity or high-affinity binding (results not shown). These findings are in agreement with several structure-activity studies for the characterization of Gal and the interaction with its receptor: it has been demonstrated that the N-terminal portion ofGal, which is completely conserved in all species, is important for receptor interaction in rat pancreatic B cells (21,34), central nervous system neurons (35,36), and isolated smooth muscle strips (14,29). In contrast, a number of Gal-specific antisera reacting with the C-terminal part of the peptide show poor cross-reactivity among species and divergent tissue levels of Gal (4, 6).…”

Section: Discussionsupporting

confidence: 92%

“…In comparison to bovine Gal (bGal) and rGal, 7 and 4 residues are different, respectively. All amino acid substitutions are restricted to the C-terminal part of the molecule (positions [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. Table 1 compares the sequences of hGal, rGal, pGal, and bGal.…”

Section: Discussionmentioning

confidence: 99%

“…Brand, Massachusetts General Hospital, Boston) expressing high numbers of Gal receptors. Cells were grown, passaged, and harvested, as described (21,22). For binding assays, 1 x 106 cells were incubated in 0.2 ml of Krebs-Ringer/bacitracin (1 g/liter) for 30 min at 250C (final volume, 0.2 ml) with 11I-labeled rGal (20,000 cpm) alone or in the presence of unlabeled rGal as standard (1 pM to 1 ,uM).…”

Section: Methodsmentioning

confidence: 99%

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Isolation and primary structure of pituitary human galanin, a 30-residue nonamidated neuropeptide.

Schmidt

Kratzin

Eckart

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Galanin (Gal), a 29-amino acid C-terminally amidated neuropeptide, is widely distributed throughout the central and peripheral nervous system. The primary structures of rat and bovine Gals were derived from the cDNA sequences of their precursors. To elucidate the structure of human Gal (hGal), we extracted 280 postmortem pituitaries in trifluoroacetic acid and purified hGal binding activity, by three successive HPLC steps, to homogeneity based on a radioreceptor assay. The primary structure of hGal was determined by automatic Edman degradation to be Gly-Trp-Thr-Leu-AsnSer-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-Ala-Val-Gly-Asn-HisArg-Ser-Phe-Ser-Asp-Lys-Asn-Gly-Leu-Thr-Ser-COOH. The structure was confirmed by plasma desorption time-of-flight mass spectrometry, revealing a mass of 3156.1. Compared to the 29-residue porcine, rat, and bovine Gals, hGal uniquely comprises 30 amino acids possessing an additional nonamidated serine residue as C terminus. The nonamidated carboxylic group at the C terminus was proven by synthesis of amidated and nonamidated hGal and by mass spectrometry after selective methylation of all free carboxylic groups. Synthetic hGal possesses full biological activity on isolated rat fundus muscle strips.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation and primary structure of pituitary human galanin, a 30-residue nonamidated neuropeptide.

Schmidt

Kratzin

Eckart

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Also, several cell biological processes in insulin-producing cells are reversed or counteracted by PTX [8]. These PTX-sensitive actions include the inhibition of insulin secretion and lowering of the cytoplasmic Ca 2+ concentration in β-cells [9] and RINm5F cells [10,11], the inhibition of cAMP production in normal islets [12] and RINm5F cells [13] and the hyperpolarization of RINm5F cells [14]. …”

Section: Introductionmentioning

confidence: 99%

Pertussis Toxin Administered Spinally Induces a Hypoglycemic Effect on Normal and Diabetic Mice

et al. 2014

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Background/Aims: To show whether intrathecal (i.t.) treatment with pertussis toxin (PTX) produces a hypoglycemic effect in ICR, db/db and streptozotocin-treated mice. Methods: The blood glucose level (BGL) was measured after i.t. treatment with PTX, AB₅ toxins and PTX subunits. Insulin or leptin levels were measured after PTX injection. The effect of PTX on the BGL was examined in adrenalectomized (ADX) mice. Glucose transporter (GLUT) levels were determined by Western blotting. Results: PTX attenuated the elevated BGL in the D-glucose-fed model in a long-term manner. Heat-labile toxin (HLT), HLT subunit B or Shiga toxin, which belong to the AB₅ toxins, administered i.t. did not affect the BGL. PTX A protomer (PTX-A) or PTX B oligomers (PTX-B) injected i.t. did not have an effect on the BGL as well. However, combined treatment with PTX-A and PTX-B subunits caused a hypoglycemic effect. The leptin level was gradually reduced by PTX for up to 6 days, without affecting the insulin level. PTX administered i.t. significantly decreased the BGL further in ADX mice. Moreover, GLUT-2 (hypothalamus and pituitary gland), GLUT-4 (muscle) and GLUT-3 (adrenal gland) expression levels were increased, whereas GLUT-1 (brain cortex, liver, muscle and spinal cord), GLUT-2 (liver) and GLUT-3 (brain cortex and pituitary gland) expression levels were decreased. Discussion: Our data suggest that PTX administered spinally produces a hypoglycemic effect in a long-term manner, and PTX-induced hypoglycemia appears to be mediated by the reduction in activity of the glucocorticoid system. Furthermore, PTX may modulate the insulin level during hypoglycemia. Among GLUTs, GLUT-4 in muscle, GLUT-2 in the liver, hypothalamus and pituitary gland as well as GLUT-1 in the adrenal gland may be responsible for PTX-induced hypoglycemia.

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“…Recently, the structure of human Gal (hGal) was elucidated (Bersani et al 1991;Evans & Shine, 1991;Schmidt et al 1991), revealing a 30 amino acid C-terminally non-amidated peptide with more than 85% structural similarity to the other mammalian Gals. Besides its inhibitory effect on cyclic AMP production in the rat insulinoma cell line RIN m5F (Amiranoff et al 1988;Gallwitz et al 1990) and its inhibition of glucose-induced insulin release (McDonald et al 1985;de Weille et al 1988), several other endocrine and neuromodulatory effects of Gal on the hypothalamus, pituitary (Murakami et al 1987;Ottlecz et al 1988;Maiter et al 1990;Merchenthaler et al 1990), adrenal gland (Rökaeus, 1987) and intestine (Muramatsu & Yanaihara, 1988;Kuwahara et al 1989;Katsoulis et al 1990) have been described. All these effects seem to be linked to a specific Gal receptor which is associated with a GTP-binding protein sensitive to pertussis toxin Servin et al 1987;Lagny-Pourmir et al 1989a).…”

Section: Introductionmentioning

confidence: 99%

Identification and biochemical characterization of the human brain galanin receptor

Walli

Schäfer²,

Morys-Wortmann³

et al. 1994

Journal of Molecular Endocrinology

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Human galanin (hGal) is an important neuro-modulator present in the brain, gastrointestinal system and the hypothalamo-pituitary axis. A specific receptor for hGal has been identified in various areas in human brain. A single class of high affinity binding sites was found on plasma membranes of the amygdala (Kd 0.23 nM, Bmax 44 fmol/mg), the hypothalamus (Kd 0.20 nM, Bmax 25 fmol/mg) and the cortex cerebri (Kd 0.11 nM, Bmax 8.2 fmol/mg). Other brain areas, i.e. cerebellum, thalamus or pons, expressed binding sites of identical high affinity in lower quantities (Bmax < 3 fmol/mg). Specific binding of 125I-labelled hGal was found to be reversible, time- and temperature-dependent and inhibited by Ca2+, Na+ and K+ ions at a concentration of 5 mM. Non-hydrolysable guanosine nucleotides potently reduced specific binding of 125-I-labelled hGal by more than 80%. Synthetic hGal analogues substituted in the N-terminal region exhibited strongly reduced binding affinity for the hGal receptor. Using 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulphonate, hGal receptors were successfully solubilized from human cortical membranes, exhibiting no significant loss of binding affinity. Affinity cross-linking to 125I-labelled hGal revealed a labelled band of approximately 60 kDa sensitive to unlabelled Gal. This putative hGal receptor is glycosylated since its molecular size was reduced after treatment with endoglycosidase F. Receptors bound to 125I-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus communis agglutinin, suggesting that receptor glycosylation involves N-acetyl glucosamine and galactose respectively.

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Galanin: Structural requirements for binding and signal transduction in RINm5F insulinoma cells

Cited by 25 publications

References 28 publications

Isolation and primary structure of pituitary human galanin, a 30-residue nonamidated neuropeptide.

Isolation and primary structure of pituitary human galanin, a 30-residue nonamidated neuropeptide.

Pertussis Toxin Administered Spinally Induces a Hypoglycemic Effect on Normal and Diabetic Mice

Identification and biochemical characterization of the human brain galanin receptor

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