Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Suen, E T; Stefanini, E.; Clement‐Cormier, Yvonne C.

doi:10.1016/0006-291x(80)91447-3

Cited by 48 publications

(17 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Likewise, a similar discrepancy between iodoacetamine and NEM effects was also reported for doparnine (8) or histamine receptors (10), and this may be attributed to a participation of the micro environment of the thiol in the accessibility or reactivity of the latter to various thiol reagents. Furthermore, to investigate the characteris tics of the inhibitory potencies of these thiol reagents, we examined the effect of tem perature on the rate of inactivation of [3H] tryptamine binding, because the pretreatment condition of these reagents (i.e., 55-min in cubation at 37'C) also corresponds to the preincubation conditions of the membranes that induce the augmentation of specific [3H]tryptamine binding.…”

Section: Discussionsupporting

confidence: 67%

Effects of protein-modifying reagents on brain tryptamine binding sites: Possible involvement of a thiol group in temperature-induced high-affinity (3H)tryptamine binding sites.

1990

View full text Add to dashboard Cite

In addition, to at least approx. 10-4 M, the inactivation properties of NEM, PCMB, PAPMA and MA, except for NBS, were temperature-dependent. Furthermore, it was revealed that the Scatchard plot of [3H]tryptamine binding in membranes pretreated with these thiol reagents conformed to a curved line, as well as in the case of the control membranes.Nonlinear regression analysis of these data showed that NEM decreased the Bmaa values of both the high and low affinity binding sites with no significant alteration in the K" values, whereas PCMB, PAPMA and MA increased only the KD value of the high affinity sites, accompanying the decrease of the Bmax values of both sites. These results indicate that the temper ature-induced high-affinity [3H]tryptamine binding molecule(s) is a thiol protein.

show abstract

Section: Discussionsupporting

confidence: 67%

Effects of protein-modifying reagents on brain tryptamine binding sites: Possible involvement of a thiol group in temperature-induced high-affinity (3H)tryptamine binding sites.

1990

View full text Add to dashboard Cite

show abstract

“…These observations imply that the D 1 -like receptor conformation through which G αq is activated requires intact, DTT-accessible, disulfide bonds, whereas the G s /AC-coupled D 1 -like conformation might be less susceptible to, or might even benefit from, a thiol-reducing environment. A number of earlier reports showed diverse effects of reducing agents on dopamine agonist and antagonist binding to dopamine receptors, and others showed that agonist stimulation of dopamine-sensitive AC could be inhibited by the thiol-alkylating reagent n-ethylmaleimide but not by DTT (38)(39)(40)(41)(42). Perhaps, these different D 1 -like "sites" are different oligomeric conformations of the known D 1 -like (D 1 /D 1A and/or D 5 /D 1B ) receptors.…”

Section: Discussionmentioning

confidence: 99%

Physicochemical Modulation of Agonist-Induced [³⁵S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D₁-Like Receptors

Panchalingam

Undie

2005

Journal of Receptors and Signal Transduction

View full text Add to dashboard Cite

Dopamine agonist-stimulated [35S]GTPgammaS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins Gi, Gs, or Gq. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D2-like/Gi), SKF38393 (D1-like/Gq, D1-like/Gs), SKF85174 (D1-like/Gs), and SKF83959 (D1-like/Gq). Dopamine and SKF38393 significantly stimulated [35S]GTPgammaS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D1-like receptor conformations that mediate agonist stimulation of Gs/adenylylcyclase may be structurally different from those that mediate Gq/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates Gs/adenylylcyclase to one that favors Gq/PLC signaling.

show abstract

“…It had been previously found-that antagonist binding to 12 receptor was inhibited by sulfhydryl reagents (22)(23)(24)(25)(26). Although most of the reagents used were hydrophobic and could have reacted with cysteines anywhere in the protein, the polar reagents Hg2+ and 5,5'-dithio(bis-2-nitrobenzoate) also inhibited binding to the D2 receptor (25,26).…”

mentioning

confidence: 99%

A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice.

Javitch

Kaback

et al. 1994

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

130

100

View full text Add to dashboard Cite

The binding site in G-protein-linked neurotramsmitter receptors is formed among their membranepning segments. Because the binding site is in the plane of the bilayer and is accessible to charged, water-soluble agonists, it must lie in a crevice open to the extracellular, aqueous medium. Information about the structure of these receptors can be obtained by identifying the residues in the membranesnning segments which face this water-filed crevice Our understanding of the molecular mechanisms of membrane receptor function depends on our knowledge of the molecular structures of receptors and on the assignment of functional roles to structural domains. High-resolution structures of membrane receptors are not yet available, but local structural information obtained by biochemical approaches, such as affinity labeling and mutagenesis, in conjunction with low-resolution structural information obtained by biophysical approaches, has provided some insight into receptor mechanisms and the potential functional roles of specific residues and regions of receptors. Affinity labeling has the advantage that the residues labeled can be inferred to be at the surface, near the residues involved in binding; however, only a few of the residues forming a binding site are likely to be affinity labeled. Mutagenesis has the advantage that any residue can be altered; however, the mutation of residues outside of a binding site can alter binding by long-range perturbation of receptor structure, thus confounding the identification of binding-site residues.The substitution by mutagenesis of cysteines for other residues is generally well tolerated. The characteristics ofthe environment surrounding a native or engineered cysteine can be inferred from the relative rates of reaction of sulfhydrylspecific reagents that differ in polarity, charge, or size. For example, hydrophilic, lipophobic reagents will react much faster with cysteines at the water-accessible surface of the protein than at the lipid/protein interface or in the protein interior. The electrostatic potential and steric constraints around cysteines can be probed with reagents of different charges and sizes.A particularly convenient arena for this approach is in ion channels, which are formed by membrane-spanning segments ofthe proteins. Among the residues in the membrane-spanning segments, only those which face the channel lumen should be water accessible. Cysteines substituted for residues in these surfaces should be accessible to small, highly polar reagents, while cysteines substituted for residues facing away from these surfaces, toward the interior ofthe protein or toward the lipid bilayer, should be inaccessible to such reagents. By determining the functional effects of charged methanethiosulfonate (MTS) derivatives on cysteine-substitution mutants, residues lining the channels of the nicotinic acetylcholine receptor (1), GABAA y-raminobutyrate receptor (2), and the cystic fibrosis transmembrane conductance regulator (3) have been identified. Covalent modification ofs...

show abstract

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Cited by 48 publications

References 15 publications

Effects of protein-modifying reagents on brain tryptamine binding sites: Possible involvement of a thiol group in temperature-induced high-affinity (3H)tryptamine binding sites.

Effects of protein-modifying reagents on brain tryptamine binding sites: Possible involvement of a thiol group in temperature-induced high-affinity (3H)tryptamine binding sites.

Physicochemical Modulation of Agonist-Induced [³⁵S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D₁-Like Receptors

A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice.

Contact Info

Product

Resources

About

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Cited by 48 publications

References 15 publications

Effects of protein-modifying reagents on brain tryptamine binding sites: Possible involvement of a thiol group in temperature-induced high-affinity (3H)tryptamine binding sites.

Effects of protein-modifying reagents on brain tryptamine binding sites: Possible involvement of a thiol group in temperature-induced high-affinity (3H)tryptamine binding sites.

Physicochemical Modulation of Agonist-Induced [35S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D1-Like Receptors

A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice.

Contact Info

Product

Resources

About

Physicochemical Modulation of Agonist-Induced [³⁵S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D₁-Like Receptors