Back to "Homepage of Judith Klein-Seetharaman"

Back to "G-protein coupled receptors"


Proteins interacting with G-protein coupled receptors

- complexity of GPCR signaling - not only via heterotrimeric G-proteins (see references in Gether (2000) review)


Cross-talk between GPCR and other Signaling pathways

- phosphorylation by GRKs (GPCR kinases) and subsequent sequestration of the receptors form the cell surface are important for tuning off signaling and switch receptor from G-protein dependent signaling to signaling cascades normally used by growth factor receptors (references in Gether (2000) review)


Sites on G protein that interact with GPCR

Lichtarge, O., H.R. Bourne, and F.E. Cohen, Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex. Proc Natl Acad Sci U S A, 1996. 93(15): p. 7507-11. see InteractionsPredict.html

Lichtarge, O., H.R. Bourne, and F.E. Cohen, An evolutionary trace method defines binding surfaces common to protein families. J Mol Biol, 1996. 257(2): p. 342-58

- prediction of rhodopsin-G protein interaction sites based on evolutionary trace ET analysis in which surfaces are selected that do not vary within functional subgroups and that form spatial clusters

- emphasis is on the G protein side, so in order to validate model, one needs to know the literature on mutagenesis of G protein


Sites on RGS that interaction with other signaling molecules of G protein signaling cascade

Sowa, M.E., W. He, T.G. Wensel, and O. Lichtarge, A regulator of G protein signaling interaction surface linked to effector specificity. Proc Natl Acad Sci U S A, 2000. 97(4): p. 1483-8.

- same as above for G protein, this paper predicts the binding sites of RGS (regulator of G protein signaling) family of proteins

- ET identifies residues important for all members of a family, therefore the residues are likely to form a general site for regulating of signaling cascades


Sites on the GPCR that interact with the G-protein

- structural elements on GPCR that interact with G protein reviewed in and reviews quoted in Gether, 2000 review

- C-II and C-III and in some receptors the proximal part of the C-term are involved in G protein coupling

- chimera work in adrenergic and muscarinic systems defined C-III as key determinant of coupling specificity among the different G protein alpha-subunits

- C-II less important for specificity, but very important for efficiency, e.g. random mutagenesis study in muscarinic rececptor coupling: Burstein, E.S., T.A. Spalding, and M.R. Brann, The second intracellular loop of the m5 muscarinic receptor is the switch which enables G-protein coupling. J Biol Chem, 1998. 273(38): p. 24322-7:

    in C-II substitutions on one side of alpha-helix III extending into CP side cause constitutive activity, while on opposite side of the helix they impair G protein activation (see also results by Yang et al., 1995, where he showed greater than 100% G protein activation for some mutants!) - first aa are important for keeping receptor in inactive conformation, the other for activation

    the AspIII.25/3.49 of the D/E R Y motif is on the same side of the helix as the residues causing constitutive activation

- reviews:

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

2.            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.

3.         Wess, J., G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. Faseb J, 1997. 11(5): p. 346-54.

4.         Wess, J., Molecular basis of receptor/G-protein-coupling selectivity. Pharmacol Ther, 1998. 80(3): p. 231-64.

5.            Dohlman, H.G., J. Thorner, M.G. Caron, and R.J. Lefkowitz, Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem, 1991. 60: p. 653-88.

6.            Strader, C.D., T.M. Fong, M.R. Tota, D. Underwood, and R.A. Dixon, Structure and function of G protein-coupled receptors. Annu Rev Biochem, 1994. 63: p. 101-32.

- speculation that opening of helix bundle of GPCR allows insertion of the alpha-subunit C-terminus into the forming cavity, which could trigger structural changes in the adjacent alpha5-helix and beta6-strand that are transmitted to the nucleotide-binding domain via the alpha5/beta6-loop, which is in the immediate vicinity of the guanine nucleotide, ref. Iiri, T., Z. Farfel, and H.R. Bourne, G-protein diseases furnish a model for the turn-on switch. Nature, 1998. 394(6688): p. 35-8

Sites on mGluR that interact with the G protein:

    - by chimera work between receptor subtypes that couple to PLC or to inhibit adenylate cyclase [Ref:  Gomeza, J., C. Joly, R. Kuhn, T. Knopfel, J. Bockaert, and J.P. Pin, The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins. J Biol Chem, 1996. 271(4): p. 2199-205.;  Pin, J.P., C. Joly, S.F. Heinemann, and J. Bockaert, Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors. Embo J, 1994. 13(2): p. 342-8.]:

            i2 loop and membrane-proximal C0termala region are responsible for PLC activation

            these regions contain several basic residues that have been proposed to form amphipathic alpha-helices, typical of G-protein coupling domains (get references above!!!)

     - distinct roles for i2 and i3 loops in selectivity of G protein coupling demonstrated by single amino acid substitutions [Francesconi, A. and R.M. Duvoisin, Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation. J Biol Chem, 1998. 273(10): p. 5615-24]:

            critical residues in i2 interaction in mGlu1a with Gs or Gq and Lys690 in i2 switches coupling to Gi

    - summary: i3 in Class A, but i2 in Class C are most important for G protein coupling [Pin, J.P. and R. Duvoisin, The metabotropic glutamate receptors: structure and functions. Neuropharmacology, 1995. 34(1): p. 1-26]  (but see above, H8 is also important both for Class A and Class C - this splitting of roles of i2/i3 is not appropriate since the CP domain is a domain made up of the different loops not just loops dangling off the helices)


A. Review of interactions with G-proteins

Gilman, A.G. (1987) G-proteins: transducers of receptor-generated signals. Annu. Rev. Biochem 56, 615-649.



G-protein independent signaling of mGluR

is reviewed in Hermans and Challiss (2001) review


Interaction with arrestins

reviewed in Palczewski, K., Structure and functions of arrestins. Protein Sci, 1994. 3(9): p. 1355-61

- sequence comparisons

- gene and splice variants

- structural information

- interaction with GPCR: 

    arrestin enhances formation of MetaII in analogous way to extra-MetaII with G protein (Schleicher, A., H. Kuhn, and K.P. Hofmann, Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II. Biochemistry, 1989. 28(4): p. 1770-5)

    CP fragments of rhodopsin do not potently inhibit the interaction between P-rhodopsin

    only interacts with rhodopsin when it is phosphorylated (Hofmann, K.P., A. Pulvermuller, J. Buczylko, P. Van Hooser, and K. Palczewski, The role of arrestin and retinoids in the regeneration pathway of rhodopsin. J Biol Chem, 1992. 267(22): p. 15701-6)

    dissociation constant MII-arrestin complex is 50nM

    binding needs less than 200ms

    heparin mimics P-rhodopsin when bound to arrestin (arrestin shows similar exposure to proteolysis, suppreses the stabilitzation of Meta II, restores arrestin-quenched phosphodiesterase activity) Palczewski, K., A. Pulvermuller, J. Buczylko, and K.P. Hofmann, Phosphorylated rhodopsin and heparin induce similar conformational changes in arrestin. J Biol Chem, 1991. 266(28): p. 18649-54



B. Role of scaffolding proteins:

also see GPCR_PDZ.html

Review: Kreienkamp (2002) Organization of GPCR signalling complexes by scaffolding proteins. Curr. Opin. Pharm. 2, 581-586.

- covers multidomain scaffolding protein-mediated interactions (often PDZ containing)

- does not cover arrestin-mediated interactions


Examples for importance of scaffolding proteins for signaling of GPCR:

1. b2-AR in kidneys

- b2-AR activates Gas, stimulating adenylate cyclase, leading to an increase in cAMP

- in kidney: cAMP-mediated activation of protein kinase A leads to inhibition of NHE3 ( a Na+/H+ exchanger)

- expected: b2-AR  agonists should inhibit NHE3

- observed: b2-AR  agonists increase NHE3

- explanation: b2-AR C-terminus binds to NHE regulatory factor (NHE-RF) via one of its PSD-95/discs large/ZO-1 (PDZ) domains

==> the interaction of the receptor with PDZ protein overrules the effect of its intracellular G-protein cascade [Ref: Hall et al. (1998) The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature 392, 626-630.]


2. insect photoreceptors [Review: Huber A (2001) Scaffolding proteins organize multi molecular protein complexes for sensory signal transduction. Eur. J. Neurosci 14, 769-776]

- InaD (inactivation no afterpotential D), consisting of 6 PDZ domains, provides scaffold for

    - protein kinase C

    - phospholipase C (PLC)

    - TRP (transient receptor potential) channel

- InaD possibly multimerizes

- InaD reduces activation and deactivation times (as measured by mutants in InaD)

- GPCR is only peripherally associated with this complex


3. Role of receptor localization [Review Edwards, S.W., C.M. Tan, and L.E. Limbird, Localization of G-protein-coupled receptors in health and disease. Trends Pharmacol Sci, 2000. 21(8): p. 304-8]

- receptor localization is a key determinant of "readiness" for activation

- LDL receptor and CFTR channel are classical examples, where mislocalization is cause for disease --> proposal that this may be the case for GPCR too, supported by two cases

    1. Retinitis pigmentosa

            C-terminal mutations

    2. Nephrogenic diabetes insipidus

            V2 receptor mutants

- Protein protein interactions that determine the steady state localization of a receptor, not only to the membrane but even to a microdomain

    - via cytoskeletal proteins, e.g. direct interaqciton has been sohwn between G proteins and tubulin [Wang, N., K. Yan, and M.M. Rasenick, Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1. J Biol Chem, 1990. 265(3): p. 1239-42]

    - multidomain cytoskeletal anchoring protein, SSTRIP, has been shown to interact directly with the C-terminus of the somatostatin sst2 receptor: [Kreienkamp, H.J., H. Zitzer, and D. Richter, Identification of proteins interacting with the rat somatostatin receptor subtype 2. J Physiol Paris, 2000. 94(3-4): p. 193-8.;   Zitzer, H., H.H. Honck, D. Bachner, D. Richter, and H.J. Kreienkamp, Somatostatin receptor interacting protein defines a novel family of multidomain proteins present in human and rodent brain. J Biol Chem, 1999. 274(46): p. 32997-3001]

- mGluR interact with Ves/Homer family via EVH1 domain (homologous to WASP). Homer interacting proteins are identical to the Shank family of PSD proteins that interact with guanylate kinase domain associated protein (GKAP_) and the PSD95 complex. Because Shank uses distinct domains to interact with GKAP and Homer, it can bridge the two proteins. GKAP-Shank is associated with NMDA receptors via PSD95 complex, thereby coordinated interaction between NMDA, mGluR, ryanodine receptors and inositol(1,4,5)triphosphate receptors --> implications for synaptic plasticity at glutamate-mediated synapses


4. Mass spec identification of protein complexes at 5-HT2C receptors: Becamel, C., G. Alonso, N. Galeotti, E. Demey, P. Jouin, C. Ullmer, A. Dumuis, J. Bockaert, and P. Marin, Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach. Embo J, 2002. 21(10): p. 2332-42

- tyrosine-kinase, iGluR and GPCR are associated with large signaling complexes, some proteins are specific to the particular complex some are not

- protein complexes important for clustering, compartmentalization (e.g. synaptic localization) and its coupling to the signaling machinery (i.e. optimization of signaling and signaling in the absence of G proteins)

- GPCR yeast-two-hybrid:

     - get refs.:

1.         Hall, R.A., R.T. Premont, C.W. Chow, J.T. Blitzer, J.A. Pitcher, A. Claing, R.H. Stoffel, L.S. Barak, S. Shenolikar, E.J. Weinman, S. Grinstein, and R.J. Lefkowitz, The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature, 1998. 392(6676): p. 626-30.

2.         Ullmer, C., K. Schmuck, A. Figge, and H. Lubbert, Cloning and characterization of MUPP1, a novel PDZ domain protein. FEBS Lett, 1998. 424(1-2): p. 63-8.

3.            Becamel, C., A. Figge, S. Poliak, A. Dumuis, E. Peles, J. Bockaert, H. Lubbert, and C. Ullmer, Interaction of serotonin 5-hydroxytryptamine type 2C receptors with PDZ10 of the multi-PDZ domain protein MUPP1. J Biol Chem, 2001. 276(16): p. 12974-82.

4.         Zitzer, H., D. Richter, and H.J. Kreienkamp, Agonist-dependent interaction of the rat somatostatin receptor subtype 2 with cortactin-binding protein 1. J Biol Chem, 1999. 274(26): p. 18153-6.

 5.   Zitzer, H., H.H. Honck, D. Bachner, D. Richter, and H.J. Kreienkamp, Somatostatin receptor interacting protein defines a novel family of multidomain proteins present in human and rodent brain. J Biol Chem, 1999. 274(46): p. 32997-3001

- GPCR mass spec: 

    - identified 15 proteins to interact with 5HT2C receptor: Becamel, C., G. Alonso, N. Galeotti, E. Demey, P. Jouin, C. Ullmer, A. Dumuis, J. Bockaert, and P. Marin, Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach. Embo J, 2002. 21(10): p. 2332-42

        Veli3; PSD95, Dlgh3 protein (MPP3); calmodulin; F-actin capping protein beta subunit (CPAZ beta) and alpha2 subunit (CPAZ alpha2); PKC teta-interacting protein PCOT; beta actin; Rik protein; dynamin 1; spectrin alpha II chain (alpha-fodrin) 

    - 5HT2C receptor contains SSV at the C-term which is a recognition motif (X-S/T-X-I/L/V) for Class I PDZ domains, and MUPP1, a multivalent PDZ protein has been identified as a binding partner by yeast-two-hybrid screen before



C. Proteins

    1. PDZ domains

    2. homer proteins

    3. Shank/ProSAP proteins


D. Big protein complexes

- linkage between mGluR and NMDA receptor by shank may be reason for extreme physical stability of the NMDA receptor complex in the postsynaptic density of excitatory synapses in the mammalian central nervous system

- complex can be isolated from brain as a >2000kDa signaling complex [references 35,36 in Kreienkamp (2002)]

- complex contains up to 100 different proteins including scaffolding (PSD-95, shank) and signal transduction (several kinases, phosphatases, regulators of small G-proteins etc.)