GPCRs represent the largest family of integral membrane proteins and were first identified as receptor proteins that couple via heterotrimeric G-proteins to regulate a vast variety of effector proteins to modulate cellular function. It is now recognized that GPCRs interact with a myriad of proteins that not only function to attenuate their signalling but also function to couple these receptors to heterotrimeric G-protein-independent signalling pathways. In addition, intracellular and transmembrane proteins associate with GPCRs and regulate their processing in the endoplasmic reticulum, trafficking to the cell surface, compartmentalization to plasma membrane microdomains, endocytosis and trafficking between intracellular membrane compartments. The present review will overview the functional consequence of b-arrestin, receptor activity-modifying proteins (RAMPS), regulators of G-protein signalling (RGS), GPCR-associated sorting proteins (GASPs), Homer, small GTPases, PSD95/Disc Large/Zona Occludens (PDZ), spinophilin, protein phosphatases, calmodulin, optineurin and Src homology 3 (SH3) containing protein interactions with GPCRs. LINKED ARTICLESThis article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue-5/issuetoc Abbreviations 5-HT2AR, serotonin receptor subtype 2; 5HT2CR, serotonin 2C receptor; a1DAR, a1D adrenergic receptor; Arf6, ADP-ribosylation factor 6; ARNO, ARF nucleotide-binding site opener; AT1R, angiotensin II type 1 receptor; b2AR, b2-adrenergic receptor; CAL, cystic fibrosis transmembrane conductance regulator-associated ligand; CASK, calcium/calmodulin-dependent serine protein kinase; CaSR, calcium sensing receptor; cGRP1/cGRP2, calcitonin gene-related peptides; CIPP, channel-interacting PDZ protein; CRF, corticotrophin-releasing factor; CRFR1, corticotropin releasing factor receptor 1; CRLR, calcitonin-like receptor; CXCR2, IL-8 receptor B; CXCR4, chemokine receptor type 4; EBP50, ERM-binding phosphoprotein 50; EEA1, effector early endosome antigen 1; EVH, ENA/VASP homology domain; GABAB, GABA type B receptor; GAPs, GTPase activating proteins; GASP, GPCR-associated sorting protein; GDP, guanosine diphosphate; GEFs, guanine nucleotide exchange factors; GIPC, GIPC PDZ domain containing family member 1; GRB2, growth factor receptor-bound protein 2; GRKs, GPCR kinases; MAGI, membrane-associated guanylate kinase inverted; MAGUK, membrane-associated guanylate kinase; mGluRs, metabotropic glutamate receptors; MINT1, Munc-18-interacting protein 1; MPP3, p55 subfamily member 3; MT1, melatonin type 1 receptor; MUPP1, multi-PDZ-domain protein; Nck, non-catalytic region of tyrosine kinase adaptor protein; NHERF, Na + /H + exchanger regulatory factor 1; NSF, N-ethylmaleimide-sensitive factor; P2Y1, P2Y1 purinergic receptors; PAK, p21-activated kinase...

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membraneassociated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na 1 /H 1 exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domaincontaining kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Ga-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and leukemia-associated RhoGEF), RGS3 and RGS12, spinophilin and neurabin-1, SRC homology 3 domain and multiple ankyrin repeat domain (Shank) proteins (Shank1, Shank2, and Shank3), partitioning defective proteins 3 and 6, multiple PDZ protein 1, Tamalin, neuronal nitric oxide synthase, syntrophins, protein interacting with protein kinase C a 1, syntenin-1, and sorting nexin 27.

Functional characterization of the GPCR interactome has been focused predominantly on intracellular interactions, yet GPCRs are increasingly found in complex with extracellular proteins. Extracellular leucine-rich repeat fibronectin type III domain containing 1 (ELFN1) was recently reported to physically anchor mGluR6 and mGluR7 across retinal and hippocampal synapses, respectively; however, the consequence of transsynaptic interactions on properties and pharmacology of these receptors are unknown. In the current study, we explore the effects of ELFN1 on mGluR signaling and pharmacology. First, we established the binding specificity of ELFN1 and found it to be recruited selectively to all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), but not other mGluR species. Using site-directed mutagenesis we mapped binding determinants of this interaction to two distinct sites on the ELFN1 ectodomain. To evaluate functional aspects of the interaction, we developed a transcellular signaling assay in reconstituted HEK293 cells which monitors changes in mGluR activity in one cell following its exposure to separate ELFN1-containing cells. Using this platform, we found that ELFN1 acts as an allosteric modulator of class III mGluR activity in suppressing cAMP accumulation: altering both agonist-induced and constitutive receptor activity. Using bioluminescence resonance energy transfer-based real-time kinetic assays, we established that ELFN1 alters the ability of mGluRs to activate G proteins. Our findings demonstrate that core properties of class III mGluRs can be altered via extracellular interactions with ELFN1 which serves as a transsynaptic allosteric modulator for these receptors. Furthermore, our unique assay platform opens avenues for exploring transcellular/transsynaptic pharmacology of other GPCR transcomplexes.

Background: CRFR1 regulates the physiological response to stress and is implicated in the manifestation of depression. Results: SAP97 interacts and co-localizes with CRFR1, suppresses CRFR1 endocytosis, and is required for CRFR1-mediated ERK1/2 phosphorylation. Conclusion: SAP97 functionally regulates CRFR1 trafficking and signaling. Significance: This is the first documentation of functional regulation of CRFR1 by a specific PDZ protein.

The functional characterization of the GPCR interactome has predominantly focused on intracellular binding partners; however, the recent emergence of trans-synaptic GPCR complexes represents an additional dimension to GPCR function that has previously been unaccounted for in drug discovery. Here, we characterize ELFN2 as a novel post-synaptic adhesion molecule with a distinct expression pattern throughout the brain and a selective binding with group III metabotropic glutamate receptors (mGluRs) in trans. Using a transcellular GPCR signaling platform, we report that ELFN2 critically alters group III mGluR secondary messenger signaling by directly altering G protein coupling kinetics and efficacy. Loss of ELFN2 in mice results in the selective downregulation of group III mGluRs and dysregulated glutamatergic synaptic transmission. Elfn2 knockout (Elfn2 KO) mice also feature a range of neuropsychiatric manifestations including seizure susceptibility, hyperactivity, and anxiety/compulsivity which can be rescued by pharmacological augmentation of group III mGluRs. Thus, we conclude that extracellular trans-synaptic scaffolding by ELFN2 in the brain is a cardinal organizational feature of group III mGluRs essential for their signaling properties and brain function.