Cross-linking of glycine receptor transmembrane segments two and three alters coupling of ligand binding with channel opening

Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. d-Tyr-Gly-[(Nle28,31,d-Trp30)CCK-26–32]-2-phenylethyl ester (d-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligand-stimulated internalization was studied morphologically using fluorescent CCK and d-Trp-OPE. d-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not d-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threonine-rich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted d-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting d-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309–323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to d-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309–323 to inhibit its d-Trp-OPE-stimulated internalization. Thus the 309–323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.

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Alpha 1 adrenoceptor activation potentiates taurine response mediated by protein kinase C in substantia nigra neurons

Journal of Neurophysiology ◽

10.1152/jn.1996.76.4.2455 ◽

1996 ◽

Vol 76 (4) ◽

pp. 2455-2460 ◽

Cited By ~ 14

Author(s):

J. Nabekura ◽

T. Omura ◽

N. Horimoto ◽

T. Ogawa ◽

N. Akaike

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Substantia Nigra ◽

Single Channel ◽

Glycine Receptor ◽

Peak Amplitude ◽

Whole Cell ◽

Kinase C ◽

Channel Opening ◽

S 100

1. The potentiation of glycine receptor-mediated taurine response (Itau) by alpha 1 adrenoceptor activation was investigated in neurons freshly dissociated from the rat substantia nigra (SN) using a nystatin perforated-patch recording. 2. Norepinephrine (NE) at a concentration of 10(-4) M in the presence of 10(-5) M yohimbine and 10(-5) M propranolol potentiated the peak amplitude of Itau (10(-3) M) at a holding potential of -40 mV under voltage clamp conditions. NE could be substituted by phenylephrine at this potentiation. 3. This potentiation of the taurine response persisted in the treatment with pertussis toxin (500 ng/ml) for 18 h. The intracellular application of GDP-beta S (100 microM) with a conventional whole cell patch recording mode abolished the effect of alpha 1 adrenoceptor activation on the Itau. 4. Staurosporine (10(-7) M) blocked the enhancement of Itau by 10(-4) M NE with 10(-5) M yohimbine and 10(-5) M propranolol. In additional phorbol-12-myristate 13-acetate (10(-5) M) potentiated Itau. 5. The intracellular application of 0.275 U/ml protein kinase C (PKC) with a conventional whole cell configuration gradually increased the peak amplitude of Itau. On the other hand, intracellular perfusion either without PKC or with PKC plus 4 microM PKC (19-36), a PKC inhibitor, did not potentiate Itau. 6. A single channel recording in a cell attached configuration revealed that NE (10(-4) M) with 10(-5) M yohimbine and 10(-5) M propranolol increased the total open time of the taurine-activated channel. This increase of the channel opening was antagonized by staurosporine (10(-7) M). 7. Neither tapsigargin (10(-6) M), LiCl (10(-4) M), trifluoperazine (10(-5) M) nor (S)-5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (10(-4) M) applied in the perfusate were found to affect the potentiation of Itau by alpha 1 adrenoceptor. The intracellular application of inositol triphosphates (10(-4) M) in a conventional whole cell recording also had no effect on Itau. 8. These findings thus indicate that alpha 1 adrenoceptor coupled with pertussis-insensitive G protein increases the intracellular PKC activity, thus leading to an increase in the channel opening activated by taurine and an enhancement of the peak amplitude of Itau in the SN neurons.

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Inhibition of Oxidative Cross-linking between Engineered Cysteine Residues at Positions 332 in Predicted Transmembrane Segments (TM) 6 and 975 in Predicted TM12 of Human P-glycoprotein by Drug Substrates

Journal of Biological Chemistry ◽

10.1074/jbc.271.44.27482 ◽

1996 ◽

Vol 271 (44) ◽

pp. 27482-27487 ◽

Cited By ~ 59

Author(s):

Tip W. Loo ◽

David M. Clarke

Keyword(s):

Cross Linking ◽

Cysteine Residues ◽

Transmembrane Segments ◽

P Glycoprotein

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Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores

Biochemical Journal ◽

10.1042/bj2770067 ◽

1991 ◽

Vol 277 (1) ◽

pp. 67-72 ◽

Cited By ~ 7

Author(s):

J J Remes ◽

U E Petäjä-Repo ◽

H J Rajaniemi

Keyword(s):

Ligand Binding ◽

Species Difference ◽

Cross Linking ◽

Complex Type ◽

High Affinity ◽

Affinity Ligand ◽

Pngase F ◽

Formyl Peptide ◽

Membrane Bound ◽

High Affinity Ligand

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.

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Complex Interaction of the MoeA-N and MoeA-C Domains of Gephyrin Is Required for the Immortalization Activity of MLL-Gephyrin.

Blood ◽

10.1182/blood.v108.11.1936.1936 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1936-1936

Author(s):

Toshihiko Imamura ◽

Roger T. Luo ◽

Michael J. Thirman

Keyword(s):

Amino Acids ◽

Glycine Receptor ◽

Cross Linking ◽

Receptor Subtypes ◽

Protein Fusion ◽

Dimerization Domain ◽

Gabaa Receptor Subtypes ◽

Colony Forming Assay ◽

Partner Proteins

Abstract More than 40 proteins have been identified as partners of MLL in acute leukemia. The partner proteins are categorized based on their subcellular localization into either the nucleus or cytoplasm. Recent studies have proposed that the mechanism of transformation of MLL-cytoplasmic protein fusion products is mediated by the oligomerization ability of partner proteins. Gephyrin, which is involved in synaptic anchoring of glycine receptor and certain GABAA receptor subtypes, is a rare partner of MLL in patients with AML. Using an in vivo cross linking assay, we confirmed the dimerization activity of a domain within the MoeA-N domain of Gephyrin, which is located at amino acids 456–476. However, we found that this domain was dispensable for immortalization in the methylcellulose colony-forming assay, indicating that the presence of a dimerization domain was not sufficient for transformation. We also observed that the Tubulin binding domain, which is located at amino acids 322–376, was also dispensable. To evaluate which domain of Gephyrin is critical for the immortalization activity of MLL-Gephyrin, we performed a colony-forming assay in methylcellulose and found that both the MoeA-N (amino acids 354–528) and the MoeA-C (amino acids 691–769) domains of Gephyrin are essential for immortalization. In addition, we observed that a MLL- Gephyrin (444–476) deletion mutant formed dimers using the in vivo cross linking assay. To explore the possibility that both MoeA-N and MoeA-C domains are associated with dimer formation of MLL- Gephyrin, we performed co-immunoprecipitation experiments with three fragments of Gephyrin, MoeA-N, MoCF (amino acids 532–690) and MoeA-C. Our data showed that both the MoeA-N and MoeA-C domains interacted with the MoCF domain of Gephyrin. Taken together, these findings show that Gephyrin has multiple dimerization domains, and that the interaction that links the MoeA-N, MoeA-C, and MoCF domains is critical for the immortalization activity of the MLL- Gephyrin fusion protein. Our data indicate that complex patterns of dimerization exist among MLL partners and suggest that specific types of dimerization domains might be critical for MLL-associated transforming activity.

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The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

The Journal of General Physiology ◽

10.1085/jgp.116.3.327 ◽

2000 ◽

Vol 116 (3) ◽

pp. 327-340 ◽

Cited By ~ 84

Author(s):

Claudio Grosman ◽

Frank N. Salamone ◽

Steven M. Sine ◽

Anthony Auerbach

Keyword(s):

Acetylcholine Receptors ◽

Single Channel ◽

Channel Gating ◽

Congenital Myasthenic Syndrome ◽

Control Element ◽

Dependent Manner ◽

Α Subunit ◽

Transmembrane Segments ◽

Channel Opening ◽

Myasthenic Syndrome

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.

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