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G-protein coupled receptor Ligand Binding

Simpson, M. M., J. A. Ballesteros, et al. (1999). "Dopamine D4/D2 receptor selectivity is determined by A divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments." Mol Pharmacol 56(6): 1116-26.

- ligand binding residues common to all types of dopamine receptors: dopamine ligand is charged, water-soluble -> binding site needs to be water-accessible

    1. electrostatic interactions between protonated amine of ligand and a conserved Asp in TM3

    2. H-bond to serines in TM5

    3. aromatic ring that interacts with the aromatic cluster in TM6

- to determine difference in pharmacological properties of subtypes of dopamine receptors:

    must reside in non-conserved sequence

- method: 

    1. identify all non-conserved residues between D2 and D4 dopamine receptors: 80

    2. which are solvent accessible (as determined by substituted cysteine accessibility method): 20 (out of 80 above)

    3. which are conserved/non-conserved: 6 conserved, 14 non-conserved

    4. replace in D2 background those 14 residues with D4 residues, and vice versa (single mutations and multiple mutations)

- result: V91F/F110L/V111M/Y408V (all are part of TM2, TM3 and TM7 aromatic microdomain) in D2 has D4-like affinity, suggesting that these four residues encode the molecular determinants for D2/D4 binding specificity

- while degree of structural similar between related GPCR is unknown, the surface of the binding-site identified in dopamine receptor may be general for Class A GPCR ligand binding sites, including positions 2.60, 2.61, 2.64, 3.28, 3.29, 7.35


Review of Ligand binding domain (Gether, 2000)

- best characterized binding sites, for retinal in rhodopsin, and for catecholamines in the adrenergic receptors

- previous reviews of ligand binding domains:     

        Kobilka, B., Adrenergic receptors as models for G protein-coupled receptors. Annu Rev Neurosci, 1992. 15: p. 87-114.

        Savarese, T.M. and C.M. Fraser, In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem J, 1992. 283 ( Pt 1): p. 1-19.

        Strader, C.D., T.M. Fong, M.P. Graziano, and M.R. Tota, The family of G-protein-coupled receptors. Faseb J, 1995. 9(9): p. 745-54.

        Ji, T.H., M. Grossmann, and I. Ji, G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem, 1998. 273(28): p. 17299-302

- agonists and antagonists of peptide receptors may have different binding sites than endogenous peptide agonists, particularly well studied for tachykinin system, the model system for peptide GPCR


A. Rhodopsin and biogenic amines

A1. Rhodopsin

    reviewed in Sakmar, T.P., Rhodopsin: a prototypical G protein-coupled receptor. Prog Nucleic Acid Res Mol Biol, 1998. 59: p. 1-34

    Schiff base with K296 VII.10/7.43

    Counterion E113, III.04/3.28

    C9 of retinal and G121, III.12/3.36

A2. Biogenic amines

    classical "small molecule transmitters" epinephrine, norepinephrine, dopamine, serotonin, histamine and acetylcholine

    residues involved in binding are in helices III, V, VI and VII, but are deeply buried in TM domain as shown by analysis of fluorescent antagonist carazolol bound to beta2-adrenergic receptor (ref. Tota, M.R. and C.D. Strader, Characterization of the binding domain of the beta-adrenergic receptor with the fluorescent antagonist carazolol. Evidence for a buried ligand binding site. J Biol Chem, 1990. 265(28): p. 16891-7.

    more details see Gether (2000) review

B. Peptide Ligands in Class A

        - more than 50 peptide hormone and neuropeptide ligands known

        - in contrast to small molecule ligand binding that mostly bind in TM domain, peptide ligand binding involves EC domain, e.g. shown by photoaffinity crosslinking of substance P analog to Met181 in E-III loop (see references 89,90 in Gether (2000)) , but mutants of NK-1 receptor show residues involved in both EC and TM domains

B1. Takykinin system

    - substance P, neurokinin A and B (act at NK-1 receptor) and neurokinin-2 (act at NK-2 receptor) and neurokinin-3 (NK-3)

    - chimeric analysis of NK-1/NK-2 and of NK-1/NK-3 recetors (exchange of EC loops!) suggests that multiple epitopes are scattered throughout the receptors and contribute to the subtype selectivity and specificity and the binding sites of the tachykinin peptides are not fully identical:

        Yokota, Y., C. Akazawa, H. Ohkubo, and S. Nakanishi, Delineation of structural domains involved in the subtype specificity of tachykinin receptors through chimeric formation of substance P/substance K receptors. Embo J, 1992. 11(10): p. 3585-91

        Gether, U., T.E. Johansen, and T.W. Schwartz, Chimeric NK1 (substance P)/NK3 (neurokinin B) receptors. Identification of domains determining the binding specificity of tachykinin agonists. J Biol Chem, 1993. 268(11): p. 7893-8

==> clear differences in the binding modes even among homologous peptides acting at homologous receptors!!

B2. Other family A peptide receptors

interactions between N-terminus and EC loops demonstrated for (see references in Gether, 2000, review):


    neuropeptide Y

    chemokines (IL8)




    complement factor C5A








- additional points of interactions in the TM domains (i.e. EC sides of TM II, III, V, VI, VII) demonstrated for

    tripeptide TRH

    tripeptide fMLP

    larger peptides: 






- the TM contacts are different from those residues that form key contacts with biogenic amines

- all residues that form contacts are on surface of binding crevice as predicted by cysteine accessibility method, supporting "high degree of structural similarity between the receptors, even though they bind chemically very different ligands"


Feighner, S.D., A.D. Howard, K. Prendergast, O.C. Palyha, D.L. Hreniuk, R. Nargund, D. Underwood, J.R. Tata, D.C. Dean, C.P. Tan, K.K. McKee, J.W. Woods, A.A. Patchett, R.G. Smith, and L.H. Van der Ploeg, Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues. Mol Endocrinol, 1998. 12(1): p. 137-45:

- antibodies against EC and CP domains to confirm orientation of GH secretagogue receptor (GHS) in membrane (the antagonist for this receptor is somatostatin)

- mutants which show that similar aa determine ligand binding site as in amine receptors


C. nonpeptide ligands for peptide receptors in family A

         problem with peptides is low bioavailability and metabolic instability, therefore goal to develop small-molecule nonpeptide compounds that are orally active and act with high potency

        - nature itself has shown the solution to this problem: enkephalins and endorphins are endogenous peptide ligands of the opioid receptors which also bind morphine and naloxone with are nonpetide exogenous ligands

    - find both agonists and antagonists that are not peptides but that mimic the action of the peptides

    - screening of chemical libraries identified such candidates: in almost all cases, there is no structural similarity between endogenous ligands and the compounds despite a competitive ode of action, ref. Schwartz, T.W., U. Gether, H. Schambye, and S.A. Hjorth, Molecular mechanism of action of non-peptide ligands for peptide receptors. Curr Pharm Design, 1995. 1: p. 325-342

C.1. Tachykinin nonpetide antagonists

- using chimera NK-1/NK-3 receptors show that CP96,345 binds differently from substance P (several chimeric exchanges that dramatically affect CP96,345 did not affect substance P), ref. Gether, U., T.E. Johansen, R.M. Snider, J.A. Lowe, 3rd, S. Nakanishi, and T.W. Schwartz, Different binding epitopes on the NK1 receptor for substance P and non-peptide antagonist. Nature, 1993. 362(6418): p. 345-8

- CP96,345 interacts with TMV and VI in different ways than substance P

- also demonstrated by fluorescent analogues of substance P (exposed to solvent) and CP 96,345 (hydrophobic environment), ref. Turcatti, G., S. Zoffmann, J.A. Lowe, 3rd, S.E. Drozda, G. Chassaing, T.W. Schwartz, and A. Chollet, Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands. J Biol Chem, 1997. 272(34): p. 21167-75

- TM crevice of CP96,345 composed of TM III, V and VI, mostly Gln165, IV.20/4.64, His197 V.05/5.39, His265 VI.17/6.52

D. Other family A receptors

- eicosanoids (leukotrienes and prostanoids) and purines bind to TM crevices

- LH/CG, FSH, TSH bind to N-term, after binding to EC domain, the N-term part of ligand undergoes conformational change leading to secondary contact with EC loops, leads to activation of receptor

- protease activated thrombin receptor: cleavage of the N-term segment by thrombin results in 33-aa long N-term that acts as tethered ligand and interacts with EC loops to activate receptor

E. Class B receptors

N-term plays key role in binding, but N-term is NOT sufficient for binding and additional interactions are found in the EC loops, but not deep into the TM domains



    pituitary adenylate cyclase-activation polypeptide (PACAP)

        there is an NMR structure of this peptide bound to its receptor: Inooka, H., T. Ohtaki, O. Kitahara, T. Ikegami, S. Endo, C. Kitada, K. Ogi, H. Onda, M. Fujino, and M. Shirakawa, Conformation of a peptide ligand bound to its G-protein coupled receptor. Nat Struct Biol, 2001. 8(2): p. 161-5

        PACAP(1-21)NH2, position 3-7 forms a beta-coil that creates a patch of hydrophobic residues that is important for receptor binding. The C-0termainl region (8-21) forms an alpha-helix that is similar to that in the micelle bound PACAP. The conformational difference between receptor vs. micelle bound peptide is limited to 1-7, consistent with two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor


    glucagon-like peptide 1



F. Class C receptors

- ligand binding dominated by N-term