Several G-protein coupled receptors, such as the ␤1-adrenergic receptor (␤1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the ␤1-AR either as a glutathione S-transferase fusion protein in biochemical ''pull-down'' assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4͞p8͞p13 (also referred to as endophilin 1͞2͞3), a SH3 domain-containing protein family, as binding partners for the ␤1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the ␤1-AR but not to that of the ␤2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of ␤1-ARs in HEK293 cells while having no effect on ␤2-ARs. Thus, our studies demonstrate a role of the SH3p4͞p8͞p13 protein family in ␤1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

The ␤ 1 -adrenergic receptor (␤ 1 AR) is the most abundant subtype of ␤-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal ␤ 1 AR-interacting proteins, we screened a rat brain cDNA library using the ␤ 1 AR carboxyl terminus (␤ 1 AR-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 ␤ 1 AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the ␤ 1 AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length ␤ 1 AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between ␤ 1 AR and PSD-95 is mediated by the last few amino acids of the ␤ 1 AR, and mutation of the ␤-Adrenergic receptors (␤ 1 AR, ␤ 2 AR, and ␤ 3 AR) 1 are heptahelical G-protein-coupled receptors that mediate physiological responses to the hormone epinephrine and the neurotransmitter norepinephrine. ␤ 1 AR and ␤ 3 AR exhibit high affinity for both epinephrine and norepinephrine, whereas ␤ 2 AR binds with high affinity only to epinephrine. The tissue distributions of the three receptors are distinct: ␤ 2 AR is highly expressed in many tissues, ␤ 3 AR is expressed at high levels only in adipose tissue, and ␤ 1 AR is expressed at high levels in the heart and brain and lower levels elsewhere (1). In the brain, ␤ 1 AR exhibits a predominantly neuronal expression pattern, whereas ␤ 2 AR is expressed mainly in glial cells (2-4). ␤ 1 AR is thus considered to be the "synaptic" ␤-adrenergic receptor, because electrophysiological experiments with specific antagonists demonstrate that ␤-adrenergic modulation of hippocampal neuronal activity exhibits a ␤ 1 AR-like pharmacological profile (5-7).Noradrenergic stimulation of synaptic ␤ 1 -adrenergic receptors is known to potently regulate memory formation and synaptic plasticity. Emotionally charged events often lead to the creation of vivid memories (8), and the formation of such emotional memories is due in large part to a surge in noradrenaline release and consequent stimulation of brain ␤-adrenergic receptors (8 -13). The powerful effects of ␤-adrenergic stimulation on memory formation correlate well with electrophysiological experiments demonstrating profound effects of ␤-adrenergic stimulation on the development of long term potentiation (LTP), an enhancement of synaptic responses that underlies some forms of memory (14). In both the hippocampus (15)(16)(17)(18)(19)(20)(21)(22) and the amygdala (23-25), two brain regions known to play key roles in the formation of emotional memories, LTP is markedly enhanced by ␤-adrenergic stimulation. This ␤-adrenergic modulation of LTP is mediated exclusively by ␤ 1 -adrenergic receptors, because hippocampal slices prepared from mice lacking ␤ 1 -adrenergic receptors do not exhibit ...

Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNAcontaining nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a subpopulation of simian virus 40 large tumor antigen with the nuclear matrix is not quantitatively altered in ATP-depleted Cos-1 cells. Nuclear GRs which are not bound to the nuclear matrix of metabolically active cells (i.e., a DNA-binding domain deletion mutant and a ␤-galactosidase chimera possessing the GR nuclear localization signal sequence) are not recruited to the matrix upon depletion of cellular ATP. Thus, it appears that ATP depletion does not expose the GR to nuclear matrix interactions which are not normally encountered in cells but merely alters the dynamics of such interactions. The dynamic association of steroid receptors with the nuclear matrix may provide a mechanism which is utilized by these regulable transcription factors to facilitate their efficient scanning of the genome.In recent years, there has been an increased appreciation for the extent to which distinct DNA, RNA, and protein components are organized within the nucleus (68). Much of the framework responsible for maintaining nuclear organization is provided for by the nuclear ...

No abstract

The in vivo characterization of a dual adenosine A(2A)/A(1) receptor antagonist in several animal models of Parkinson's disease is described. Discovery and scale-up syntheses of compound 1 are described in detail, highlighting optimization steps that increased the overall yield of 1 from 10.0% to 30.5%. Compound 1 is a potent A(2A)/A(1) receptor antagonist in vitro (A(2A) K(i) = 4.1 nM; A(1) K(i) = 17.0 nM) that has excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse model of reserpine-induced akinesia, rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation, and MPTP-treated non-human primate model.

All steroid receptors possess a bipartite nuclear localization signal sequence (NLS) that localizes within the second zinc finger of their DNA-binding domain. Fine-structure mapping of the rat glucocorticoid receptor (rGS) NLS identified a composite signal composed of three distinct proto-NLSs that function effectively when present in unique pairs. At least one of the rGR proto-NLSs appears to influence receptor trafficking within the nucleus, as revealed by a unique nuclear staining pattern of receptors possessing a point mutation (i.e., arginine at position 496; R496), at proto-NLS, pNLS-2. Specifically, carboxyl-terminal-truncated rGRs possessing various point mutations at R496 localized within a limited number of large foci in nuclei of transiently transfected COS-1 cells. R496 mutations did not affect subnuclear targeting when present in full-length rGR, reflecting a protective effect of the receptor's ligand-binding domain that can be exerted in cis and in trans. The effects of rGR R496 mutations on subnuclear targeting were not autonomous because we also observed a coincident localization of hsp70, the 70-kDa heat shock protein, within nuclear foci that include r496 mutant receptors. The elimination of R496 mistargeting by overexpression of an hsp70 partner (i.e., the DnaJ homologue, HDJ-2/HSDJ) suggests that the hsp70/DnaJ chaperone system is mobilized to specific sites within the nucleus in response to inappropriate targeting or folding of specific mutant receptors. HDJ-2/HSDJ overexpression also corrects defective transactivation and transrepression activity of R496 mutant GRs. Thus, molecular chaperones, such as members of the hsp70 and DnaJ families, may survey the nucleus for misfolded proteins and actively participate in their refolding into biologically active conformational states.

No abstract

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.