Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

Seven-transmembrane receptors.

PDZ domains are protein-interaction domains that are often found in multi-domain scaffolding proteins. PDZ-containing scaffolds assemble specific proteins into large molecular complexes at defined locations in the cell. In the postsynaptic density of neuronal excitatory synapses, PDZ proteins such as PSD-95 organize glutamate receptors and their associated signalling proteins and determine the size and strength of synapses. PDZ scaffolds also function in the dynamic trafficking of synaptic proteins by assembling cargo complexes for transport by molecular motors. As key organizers that control synaptic protein composition and structure, PDZ scaffolds are themselves highly regulated by synthesis and degradation, subcellular distribution and post-translational modification.

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PDZ domain proteins of synapses

G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors. These receptors are natural allosteric proteins because agonist-mediated signaling by GPCRs requires a conformational change in the receptor protein transmitted between two topographically distinct binding sites, one for the agonist and another for the G protein. It is now becoming increasingly recognized, however, that the agonist-bound GPCR can also form ternary complexes with other ligands or “accessory” proteins and display altered binding and/or signaling properties in relation to the binary agonist-receptor complex. Allosteric sites on GPCRs represent novel drug targets because allosteric modulators possess a number of theoretical advantages over classic orthosteric ligands, such as a ceiling level to the allosteric effect and a potential for greater GPCR subtype-selectivity. Because of the noncompetitive nature of allosteric phenomena, the detection and quantification of such effects often relies on a combination of equilibrium binding, nonequilibrium kinetic, and functional signaling assays. This review discusses the development and properties of allosteric receptor models for GPCRs and the detection and quantification of allosteric effects. Moreover, we provide an overview of the current knowledge regarding the location of possible allosteric sites on GPCRs and candidate endogenous allosteric modulators. Finally, we discuss the potential for allosteric effects arising from the formation of GPCR oligomers or GPCRs complexed with accessory cellular proteins. It is proposed that the study of allosteric phenomena will become of progressively greater import to the drug discovery process due to the advent of newer and more sensitive GPCR screening technologies.

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G Protein-Coupled Receptor Allosterism and Complexing

Lipid rafts — specialized plasma membrane microdomains that are thought to regulate various signalling events — are the focus of intensive research into their roles in the nervous system. Here, Rasenick and colleagues review the evidence for their involvement in regulating neurotransmitter signalling. Lipid rafts are specialized structures on the plasma membrane that have an altered lipid composition as well as links to the cytoskeleton. It has been proposed that these structures are membrane domains in which neurotransmitter signalling might occur through a clustering of receptors and components of receptor-activated signalling cascades. The localization of these proteins in lipid rafts, which is affected by the cytoskeleton, also influences the potency and efficacy of neurotransmitter receptors and transporters. The effect of lipid rafts on neurotransmitter signalling has also been implicated in neurological and psychiatric diseases.

Lipid raft microdomains and neurotransmitter signalling

The endocytic pathway tightly controls the activity of G protein-coupled receptors (GPCRs). Ligand-induced endocytosis can drive receptors into divergent lysosomal and recycling pathways, producing essentially opposite effects on the strength and duration of cellular signaling via heterotrimeric G proteins, and may also promote distinct signaling events from intracellular membranes. This chapter reviews recent developments toward understanding the molecular machinery and functional implications of GPCR sorting in the endocytic pathway, focusing on mammalian GPCRs whose ligand-induced endocytosis is mediated primarily by clathrin-coated pits. Lysosomal sorting of a number of GPCRs occurs via a highly conserved mechanism requiring covalent tagging of receptors with ubiquitin. There is increasing evidence that additional, noncovalent mechanisms control the sorting of endocytosed GPCRs to lysosomes in mammalian cells. Recycling of several GPCRs to the plasma membrane is also specifically sorted, via a mechanism requiring both receptor-specific and shared sorting proteins. The current data reveal an unprecedented degree of specificity and plasticity in the cellular regulation of mammalian GPCRs by endocytic membrane trafficking. These developments have fundamental implications for GPCR pharmacology, and suggest new mechanisms that could be exploited in GPCR-directed pharmacotherapy.

Regulation of GPCRs by Endocytic Membrane Trafficking and Its Potential Implications

β1-Adrenergic Receptor Association with PSD-95 INHIBITION OF RECEPTOR INTERNALIZATION AND FACILITATION OF β1-ADRENERGIC RECEPTOR INTERACTION WITHN-METHYL-d-ASPARTATE RECEPTORS

Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediates β-1 Adrenergic Receptor Endocytosis through Different Pathways

The b1-adrenergic receptor (b1AR) is the most abundant subtype of b-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal b1AR-interacting proteins, we screened a rat brain cDNA library using the b1AR carboxyl terminus (b1AR-CT) as bait in the yeast two-hybrid system. These screens identified PSD-95, a multiple PDZ domain-containing scaffolding protein, as a specific binding partner of the b1AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the b1AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length b1AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between b1AR and PSD-95 is mediated by the last few amino acids of the b1AR, and mutation of the b1AR carboxyl terminus eliminated the binding and disrupted the co-localization of the b1AR and PSD-95 in cells. Agonist-induced internalization of the b1AR in HEK-293 cells was markedly attenuated by PSD-95 co-expression, whereas co-expression of PSD-95 has no significant effect on either desensitization of the b1AR or b1AR-induced cAMP accumulation. Furthermore, PSD-95 facilitated the formation of a complex between the b1AR and N-methyl-D-aspartate receptors, as assessed by co-immunoprecipitation. These data reveal that PSD-95 is a specific b1AR binding partner that modulates b1AR function and facilitates physical association of the b1AR with synaptic proteins, such as the N-methyl-Daspartate receptors, which are known to be regulated by b1AR stimulation.

INHIBITION OF RECEPTOR INTERNALIZATION AND FACILITATION OF b1-ADRENERGIC RECEPTOR INTERACTION WITH N-METHYL-D-ASPARTATE RECEPTORS*

1 -adrenergic receptor association with PSD-95: inhibition of receptor internalization and facilitation of 1-adrenergic receptor interaction with NMDA receptors

Mei Cong

Papers

β1-Adrenergic Receptor Association with PSD-95 INHIBITION OF RECEPTOR INTERNALIZATION AND FACILITATION OF β1-ADRENERGIC RECEPTOR INTERACTION WITHN-METHYL-d-ASPARTATE RECEPTORS

Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediates β-1 Adrenergic Receptor Endocytosis through Different Pathways

INHIBITION OF RECEPTOR INTERNALIZATION AND FACILITATION OF b1-ADRENERGIC RECEPTOR INTERACTION WITH N-METHYL-D-ASPARTATE RECEPTORS*

1 -adrenergic receptor association with PSD-95: inhibition of receptor internalization and facilitation of 1-adrenergic receptor interaction with NMDA receptors