Biomedical subjects

Lee E Limbird

Publications and source records attributed to Lee E Limbird.

17 recordsLinked to original sources

PubMed2006

Regulation of alpha2AR trafficking and signaling by interacting proteins.

The continuing discovery of new G protein-coupled receptor (GPCR) interacting proteins and clarification of the functional consequences of these interactions has revealed multiple roles for these events. Some of these interactions serve to scaffold GPCRs to particular cellular micro-compartments or to tether them to defined signaling molecules, while other GPCR-protein interactions control GPCR trafficking and the kinetics of GPCR-mediated signaling transduction. This review provides a general overview of the variety of GPCR-protein interactions reported to date, and then focuses on one prototypical GPCR, the alpha(2)AR, and the in vitro and in vivo significance of its reciprocal interactions with arrestin and spinophilin. It seems appropriate to recognize the life and career of Arthur Hancock with a summary of studies that both affirm and surprise our preconceived notions of how nature is designed, as his career-long efforts similarly affirmed the complexity of human biology and attempted to surprise pathological changes in that biology with novel, discovery-based therapeutic interventions. Dr. Hancock's love of life, of family, and of commitment to making the world a better place are a model of the life well lived, and truly missed by those who were privileged to know, and thus love, him.

14-3-3 Proteins↗

PubMed2006

Adenylyl cyclase isoform-selective regulation of vascular smooth muscle proliferation and cytoskeletal reorganization.

Compartmentation of cAMP signaling been demonstrated to be attributable to the structural association of protein kinase A (PKA) (via association with A-kinase anchoring proteins [AKAPs]) with phosphodiesterase and AKAP-dependent effector molecules. However, other mechanisms contributing to compartmentalization have not been rigorously explored, including the possibility that different isoforms of adenylyl cyclase (AC) may be functionally "compartmentalized" because of differential association with tethering or signaling molecules. To this end, we examined the effect of adenoviral transduction of representative AC isoforms (AC1, AC2, AC5, and AC6) on cellular cAMP production, PKA activation, extracellular signal-regulated kinase (ERK) activation, cell doubling and proliferation, as well as arborization responses (an index of cAMP-mediated cytoskeletal re-organization) in vascular smooth muscle cells. When isoforms were expressed at levels to achieve comparable forskolin-stimulated AC activity, only gene transfer of AC6 significantly enhanced PKA-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and arborization responses. Treatment of control cells, which express AC6 endogenously, as well as vascular smooth overexpressing the AC6 isoform with small interfering RNA directed against AC6, significantly suppressed both isoproterenol-stimulated cAMP accumulation and arborization. Notably, the selective effects of AC6 expression were abrogated in the presence of phosphodiesterase suppression. In contrast, only the expression of AC1 enhanced forskolin-stimulated association of ERK with AC, demonstrated by coimmuno-isolation of ERK with Flag-tagged AC1, but not with Flag-tagged AC6. To determine whether these isoform-selective effects of AC were unique to differentiated and morphologically compartmentalized vascular smooth muscle cells or were a general property of these isoforms, we examined the consequence of expression of these various isoforms in human embryonic kidney (HEK) cells. Indeed, we observed similar isoform-dependent association of AC1 with ERK, activation of ERK by stimulation of AC1 with forskolin, and AC1-dependent lengthening of doubling time, indicating that these properties of AC1 are cell autologous and likely result from AC1-dependent protein-protein interactions. In aggregate, these findings suggest that isoform-selective signaling complexes likely contribute to various functional consequences of cAMP elevation in vascular smooth muscle cells.

1-Methyl-3-isobutylxanthine↗

PubMed2006

Arrestin serves as a molecular switch, linking endogenous alpha2-adrenergic receptor to SRC-dependent, but not SRC-independent, ERK activation.

Our previous studies have demonstrated that neither receptor endocytosis nor arrestin is required for ERK activation by the alpha2-adrenergic receptor (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940-1944). The present studies address whether arrestin plays a role in determining the route of alpha2AR-evoked ERK signaling activation, taking advantage of endogenous expression of the alpha(2A)AR subtype in mouse embryonic fibroblasts (MEFs) and the availability of MEFs without arrestin expression (derived from Arr2,3-/- mice). Our data demonstrate that the endogenous alpha(2A)AR evokes ERK phosphorylation through both a Src-dependent and a Src-independent pathway, both of which are G protein dependent and converge on the Ras-Raf-MEK pathway. Arrestin is essential to recruit Src to this process, as alpha(2A)AR-mediated ERK signaling in Arr2,3-/- MEFs does not involve Src. Stimulation of alpha(2A)AR enhances arrestin-Src interaction and promotes activation of Src. alpha2 agonists have similar potencies in stimulating Src-dependent and Src-independent ERK phosphorylation in wild-type and Arr2,3-/- cells, respectively. However, Src-independent alpha(2A)AR-mediated ERK stimulation has both a longer duration of activation and a more rapid translocation of pERK into the nucleus when compared with Src-dependent activation. These data not only affirm the role of arrestin as an escort for signaling molecules such as Src family kinases but also demonstrate the impact of arrestin-dependent modulation on both the temporal and spatial properties of ERK activation.

Animals↗

PubMed2005

Alpha 2-adrenergic agonist enrichment of spinophilin at the cell surface involves beta gamma subunits of Gi proteins and is preferentially induced by the alpha 2A-subtype.

Agonist activation regulates reciprocal interactions of spinophilin and arrestin with the alpha2A- and alpha2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the alpha2A- and alpha2B -AR subtypes also led to spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged spinophilin examined spinophilin localization and its regulation by alpha2 -AR agonist. Agonist activation of alpha2A-AR preferentially, compared with the alpha2B-AR, led to spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from spinophilin null mice. Activation of the delta LEESSSS alpha2A-AR, which has enriched association with spinophilin compared with the wild-type (WT) alpha2A-AR, does not show an enhanced redistribution of spinophilin to the surface compared with WT alpha2A-AR, demonstrating that the ability or affinity of the receptor in binding spinophilin may be independent of the ability of the receptor to effect spinophilin redistribution to the surface. Agonist-evoked enrichment of spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of spinophilin redistribution in cells expressing the beta-adrenergic receptor kinase-C tail, which sequesters beta gamma subunits of G proteins. Together, the data suggest that agonist-evoked spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the spinophilin molecule.

Adrenergic alpha-2 Receptor Agonists↗

PubMed2005

Proteomic exploration of pancreatic islets in mice null for the alpha2A adrenergic receptor.

The present studies extend recent findings that mice null for the alpha(2A) adrenergic receptor (alpha(2A) AR KO mice) lack suppression of exogenous secretagogue-stimulated insulin secretion in response to alpha(2) AR agonists by evaluating the endogenous secretagogue, glucose, ex vivo, and providing in vivo data that baseline insulin levels are elevated and baseline glucose levels are decreased in alpha(2A) AR KO mice. These latter findings reveal that the alpha(2A) AR subtype regulates glucose-stimulated insulin release in response to endogenous catecholamines in vivo. The changes in alpha(2A) AR responsiveness and resultant changes in insulin/glucose homeostasis encouraged us to utilize proteomics strategies to identify possible alpha(2A) AR downstream signaling molecules or other resultant changes due to perturbation of alpha(2A) AR expression. Although agonist stimulation of islets from wild type (WT) mice did not significantly alter islet protein profiles, several proteins were enriched in islets from alpha(2A) AR KO mice when compared with those from WT mice, including an enzyme participating in insulin protein processing. The present studies document the important role of the alpha(2A) AR subtype in tonic suppression of insulin release in response to endogenous catecholamines as well as exogenous alpha(2) agonists and provide insights into pleiotropic changes that result from loss of alpha(2A) AR expression and tonic suppression of insulin release.

Adrenergic alpha-2 Receptor Agonists↗

PubMed2004

Calmodulin interacts with the V2 vasopressin receptor: elimination of binding to the C terminus also eliminates arginine vasopressin-stimulated elevation of intracellular calcium.

To identify molecules that might contribute to V2 vasopressin receptor (V2R) trafficking or signaling, we searched for novel interacting proteins with this receptor. Preliminary data, using the V2R C terminus as bait in a yeast two-hybrid screen, revealed calmodulin as a binding partner. Because calmodulin interacts with other G protein-coupled receptors, we explored this interaction and its possible functional relevance in greater detail. A Ca2+ -dependent interaction occurs between calmodulin-linked agarose and the holo-V2R as well as the V2R C terminus. Truncation and site-directed mutagenesis of the V2R C terminus revealed an involvement of an RGR sequence in this interaction. NMR studies showed that a peptide fragment of the V2R C terminus containing the RGR sequence binds to calmodulin in a Ca2+ -dependent manner with a Kd < or =1.5 microm; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of approximately 200 nm for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells.

Amino Acid Sequence↗

PubMed2004

Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors.

Arrestin regulates almost all G protein-coupled receptor (GPCR)-mediated signaling and trafficking. We report that the multidomain protein, spinophilin, antagonizes these multiple arrestin functions. Through blocking G protein receptor kinase 2 (GRK2) association with receptor-Gbetagamma complexes, spinophilin reduces arrestin-stabilized receptor phosphorylation, receptor endocytosis, and the acceleration of mitogen-activated protein kinase (MAPK) activity following endocytosis. Spinophilin knockout mice were more sensitive than wild-type mice to sedation elicited by stimulation of alpha2 adrenergic receptors, whereas arrestin 3 knockout mice were more resistant, indicating that the signal-promoting, rather than the signal-terminating, roles of arrestin are more important for certain response pathways. The reciprocal interactions of GPCRs with spinophilin and arrestin represent a regulatory mechanism for fine-tuning complex receptor-orchestrated cell signaling and responses.

Adenosine↗

PubMed2004

The receptor concept: a continuing evolution.

This review briefly summarizes the development of the receptor concept, the identification of receptors based first on biological response data and subsequently on radioligand binding properties, and the biological and physiological understandings that these approaches have made possible. The development of receptor characterization began with receptors that ultimately were discovered to mediate response by coupling to G-binding proteins, also known as G protein-coupled receptors (GPCRs). Consequently, many if not all of the examples in this overview will describe studies characterizing GPCRs in general, and adrenergic receptors in particular. The purpose of this review, however, is not a detailed chronological account of a huge literature, but rather an overview of the fundamental questions posed and answered by these studies.

Animals↗

PubMed2004

Membrane trafficking of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) modulate diverse physiological and behavioral signaling pathways by virtue of changes in receptor activation and inactivation states. Functional changes in receptor properties include dynamic interactions with regulatory molecules and trafficking to various cellular compartments at various stages of the life cycle of a GPCR. This review focuses on trafficking of GPCRs to the cell surface, stabilization there, and agonist-regulated turnover. GPCR interactions with a variety of newly revealed partners also are reviewed with the intention of provoking further analysis of the relevance of these interactions in GPCR trafficking, signaling, or both. The disease consequences of mislocalization of GPCRs also are described.

Animals↗

PubMed2004

Study of G-protein-coupled receptor-protein interactions using gel overlay assays and glutathione-S-transferase-fusion protein pull-downs.

Numerous recent studies have suggested that the predicted cytosolic domains of G-protein-coupled receptors (GPCRs) represent a surface for association with proteins that may serve multiple roles in receptor localization, turnover, and signaling beyond the well-characterized interactions of these receptors with heterotrimeric G-proteins. This chapter describes two in vitro methods for ascertaining interactions between GPCRs and various binding partners: gel overlay strategies and GST-fusion protein pull-downs.

Animals↗

PubMed2003

Appropriate polarization following pharmacological rescue of V2 vasopressin receptors encoded by X-linked nephrogenic diabetes insipidus alleles involves a conformation of the receptor that also attains mature glycosylation.

To understand the mechanisms of G protein-coupled receptor delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2Rs) in polarized Madin-Darby canine kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates arginine vasopressin (AVP)-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, Delta V278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of Delta V278 V2R, parallels acquisition of AVP-stimulated cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized epithelial cells.

Amino Acid Substitution↗

PubMed2003

Spinophilin stabilizes cell surface expression of alpha 2B-adrenergic receptors.

The third intracellular (3i) loops of the alpha 2A- and alpha 2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha 2A, alpha 2B, and alpha 2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha 2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha 2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha 2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2) < or = 60 min) apical versus slower (t(1/2) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 h and eliminated the rapid phase (0-60 min) of alpha 2B-AR turnover on that surface. Furthermore, we examined alpha 2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha 2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha 2B-AR and presumably all three alpha2-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha 2-ARs to proteins interacting with spinophilin via other domains.

Adrenergic alpha-Agonists↗

PubMed2002

Regulated interactions of the alpha 2A adrenergic receptor with spinophilin, 14-3-3zeta, and arrestin 3.

The present studies demonstrate that no single stretch of sequence in the third intracellular (3i) loop of the alpha(2A) adrenergic receptor (alpha(2A)-AR) can fully account for its previously described interactions with spinophilin (Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003-15008), 14-3-3zeta (Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462-13469), and arrestin 3 (Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842), suggesting that a three-dimensional surface, rather than a linear sequence, provides the basis for these interactions as proposed for 3i loop tethering of the alpha(2A)-AR to the basolateral surface of Madin-Darby canine kidney cells (Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 16331-16336). Sequences at the extreme N-terminal and C-terminal ends of the 3i loop are critical for interaction with spinophilin but not for interaction with 14-3-3zeta or arrestin 3, for which the C-terminal half of the loop is more important. Competition binding for (35)S-labeled alpha(2A)-AR 3i loop binding to glutathione S-transferase (GST)-spinophilin amino acids 151-444 revealed a relative affinity of spinophilin congruent with arrestin > 14-3-3zeta for the unphosphorylated alpha(2A)-AR 3i loop. Agonist occupancy of the alpha(2A)-AR increases receptor association with spinophilin, and arrestin 3 appears to compete for this enrichment. However, when the G protein-coupled receptor kinase 2 substrate sequence was deleted from the 3i loop, arrestin 3 could not compete for the agonist-enriched binding of spinophilin to the mutant alpha(2A)-AR. These data are consistent with a model where sequential or competitive interactions among spinophilin, arrestin, and/or 14-3-3zeta play a role in alpha(2A)-AR functions.

Amino Acid Substitution↗

PubMed2002

Mutation of the alpha2A-adrenoceptor impairs working memory performance and annuls cognitive enhancement by guanfacine.

Norepinephrine strengthens the working memory, behavioral inhibition, and attentional functions of the prefrontal cortex through actions at postsynaptic alpha2-adrenoceptors (alpha2-AR). The alpha2-AR agonist guanfacine enhances prefrontal cortical functions in rats, monkeys, and human beings and ameliorates prefrontal cortical deficits in patients with attention deficit hyperactivity disorder. The present study examined the subtype of alpha2-AR underlying these beneficial effects. Because there are no selective alpha2A-AR, alpha2B-AR, or alpha2C-AR agonists or antagonists, genetically altered mice were used to identify the molecular target of the action of guanfacine. Mice with a point mutation of the alpha2A-AR, which serves as a functional knock-out, were compared with wild-type animals and with previously published studies of alpha2C-AR knock-out mice (Tanila et al., 1999). Mice were adapted to handling on a T maze and trained on either a spatial delayed alternation task that is sensitive to prefrontal cortical damage or a spatial discrimination control task with similar motor and motivational demands but no dependence on prefrontal cortex. The effects of guanfacine on performance of the delayed alternation task were assessed in additional groups of wild-type versus alpha2A-AR mutant mice. We observed that functional loss of the alpha2A-AR subtype, unlike knock-out of the alpha2C-AR subtype, weakened performance of the prefrontal cortical task without affecting learning and resulted in loss of the beneficial response to guanfacine. These data demonstrate the importance of alpha2A-AR subtype stimulation for the cognitive functions of the prefrontal cortex and identify the molecular substrate for guanfacine and novel therapeutic interventions.

Adrenergic alpha-2 Receptor Agonists↗

PubMed2002

Heterozygous alpha 2A-adrenergic receptor mice unveil unique therapeutic benefits of partial agonists.

Genetic manipulation of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) in mice has revealed the role of this subtype in numerous responses, including agonist-induced hypotension and sedation. Unexpectedly, alpha(2)-agonist treatment of mice heterozygous for the alpha(2A)-AR (alpha(2A)-AR(+/-)) lowers blood pressure without sedation, indicating that more than 50% of alpha(2A)-AR must be activated to evoke sedation. We postulated that partial activation of alpha(2A)-AR in wild-type alpha(2A)-AR(+/+) animals could be achieved with partial agonists, agents with variable ability to couple receptor occupancy to effector activation, and might elicit one versus another pharmacological response. In vitro assays reveal that moxonidine is a partial agonist at alpha(2A)-AR. Although moxonidine was developed to preferentially interact with imidazoline binding sites, it requires the alpha(2A)-AR to lower blood pressure because we observe no hypotensive response to moxonidine in alpha(2A)-AR-null (alpha(2A)-AR(-/-)) mice. Moreover, we observe that moxonidine lowers blood pressure without sedation in wild-type mice, consistent with the above hypothesis regarding partial agonists. Our findings suggest that weak partial agonists can evoke response-selective pathways and might be exploited successfully to achieve alpha(2A)-AR pharmacotherapy where concomitant sedation is undesirable, i.e., in treatment of depression or attention deficit hyperactivity disorder, in suppression of epileptogenesis, or enhancement of cognition. Furthermore, rigorous physiological and behavioral assessment of mice heterozygous for particular receptors provides a general strategy for elucidation of pathways that might be selectively activated by partial agonists, thus achieving response-specific therapy.

Adrenergic alpha-Agonists↗

PubMed2002

Morphological and biochemical strategies for monitoring trafficking of epitope-tagged G protein-coupled receptors in agonist-naive and agonist-occupied states.

Epitope tagged alpha 2-AR subtypes have been used to address a variety of cell biological questions, and the strategies used are readily applicable to all GPCR as well as other cell surface proteins. We have provided detailed protocols for successful utilization of the epitope-tagged receptor in the studies of protein localization and trafficking in epithelial cells, and the mechanisms by which this is achieved. We have also described reversible biotinytion strategies to examine agonist-dependent (and independent) receptor turnover at the cell surface.

Amino Acid Sequence↗

PubMed2002

G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction.

The mechanism by which G protein-coupled receptors (GPCRs) translate extracellular signals into cellular changes initially was envisioned as a simple linear model: activation of the receptor by agonist binding leads to dissociation of the heterotrimeric GTP-binding G protein into its alpha and betagamma subunits, both of which can activate or inhibit various downstream effector molecules. The plethora of recently described multidomain scaffolding proteins and accessory/chaperone molecules that interact with GPCR, including GPCR themselves as homo- or heterodimers, provides for diverse molecular mechanisms for ligand recognition, signalling specificity, and receptor trafficking. This review will summarize the recently described GPCR-interacting proteins and their individual functional roles, as understood. Implicit in the search for the functional relevance of these interactions is the expectation that enhancement or disruption of target cell-specific events could serve as highly selective therapeutic opportunities.

Cell Membrane↗