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Biomedical subjects

L E Limbird

Publications and source records attributed to L E Limbird.

At least 37 records · Page 2Linked to original sources
PubMed1998

A point mutation (D79N) of the alpha2A adrenergic receptor abolishes the antiepileptogenic action of endogenous norepinephrine.

Norepinephrine serves as a neurotransmitter for a population of neurons the cell bodies of which reside in a brainstem nucleus and the axons of which project widely to discrete subsets of forebrain neurons. Norepinephrine powerfully inhibits epileptogenesis in the kindling model. Pharmacological methods have demonstrated that the antiepileptogenic actions of norepinephrine are exerted via alpha2 adrenergic receptors residing on targets of noradrenergic neurons. The existence of three alpha2 adrenergic receptor subtypes together with the lack of subtype-specific ligands has precluded understanding the role of individual alpha2 adrenergic receptor subtypes in the antiepileptogenic actions of norepinephrine. Gene targeting was used to introduce a point mutation into the alpha2A adrenergic subtype in the mouse genome. The mutation produced a marked enhancement of epileptogenesis and abolished the proepileptogenic actions of the alpha2 adrenergic receptor antagonist idazoxan. These studies reveal the crucial contribution of the alpha2A receptor subtype in suppression of epileptogenesis. Development of agents that promote selective activation of the alpha2A receptor subtype may provide novel therapeutic strategies for the prophylaxis of epilepsy.

Adrenergic alpha-Agonists↗
PubMed1998

Trafficking itineraries of G protein-coupled receptors in epithelial cells do not predict receptor localization in neurons.

These studies explored whether the localization and differential trafficking itineraries of G protein-coupled receptors in polarized renal epithelial cells might predict their localization in neurons, as suggested previously. The A1 adenosine receptor is preferentially localized apically, whereas the three alpha 2-adrenergic receptor (alpha 2 AR) subtypes are localized on the lateral subdomain of Madin-Darby canine kidney cells. The alpha 2A AR and alpha 2C AR subtypes achieve this localization by direct targeting; alpha 2B AR is randomly delivered but preferentially retained basolaterally. Despite their differing itineraries in renal epithelial cells, all three epitope-tagged alpha 2-adrenergic receptor subtypes were found in neuronal cell bodies and along the entire length of the neuronal processes following transfection into long term primary cultures of mouse embryonic spinal cord neurons. In a small fraction of neuronal cells, expression of A1 adenosine receptor was limited to a short segment of their processes, otherwise it too was distributed in the soma and neuronal processes. A mutant alpha 2A AR that exhibits an accelerated turnover on the surface of epithelial cells nonetheless has a localization pattern indistinguishable from the wild-type alpha 2A AR in spinal cord neurons. Thus, unlike examples for GPI-anchored apical proteins that distribute along axons or single transmembrane-spanning basolateral proteins that localize to the soma of neurons, similar predictions do not appear to apply for polytopic G protein-coupled receptors.

Animals↗
PubMed1998

Evaluation of the alpha(2A)-adrenergic receptor gene in a heritable form of temporal lobe epilepsy.

An autosomal dominant form of human temporal lobe epilepsy (TLE) has been mapped to a region of chromosome 10q that contains the intronless alpha(2A)-adrenergic receptor (alpha(2A)AR) gene. Because mutation of the alpha(2A)AR gene in the mouse fosters epileptogenesis, we developed methods for analysis of the alpha(2A)AR coding region applicable to any pathophysiologic state in which the alpha(2A)AR could be implicated in the disease mechanism. This study rules out mutations in the alpha(2A)AR coding region as causal for this form of autosomal dominant TLE.

Animals↗
PubMed1998

Alpha 2-adrenergic receptor subtypes: subtle mutation of the alpha 2A-adrenergic receptor in vivo by gene targeting strategies reveals the role of this subtype in multiple physiological settings.

Alpha 2-adrenergic receptors (alpha 2AARs) are coupled by pertussis-toxin sensitive G proteins to various effectors, including adenylyl cyclase and ion channels. The alpha 2AARs respond to endogenous norepinephrine and epinephrine to elicit a variety of physiological responses, including inhibition of neurotransmitter release, suppression of insulin release from pancreatic beta cells, activation of platelet aggregation, and contraction of arteriolar smooth muscle. Three distinct alpha 2AR subtypes (alpha 2A, alpha 2B, alpha 2C) have been characterized by both pharmacological and molecular biological approaches; however, the lack of subtype-specific ligands has precluded an understanding of the physiological relevance of each subtype. Previous studies demonstrated that mutation of a conserved aspartate residue in the alpha 2AAR to asparagine (D79N alpha 2AAR) resulted in a receptor that retained its ability to inhibit voltage-gated Ca2+ channels and cAMP production but was unable to activate K+ currents in AtT20 cells (Surprenant et al., 1992). To explore the physiological role of the alpha 2AAR subtype and to evaluate the selectivity of alpha 2AAR effects with respect to various signal transduction pathways, we used gene targeting in embryonic stem cells to create a mouse line that expresses the mutant D79N alpha 2AAR instead of the wild-type alpha 2AAR. We established a D79N alpha 2AAR mouse line and characterized various alpha 2AAR-mediated physiological functions in these mutant mice. Because the in vivo D79N alpha 2AAR is expressed at a reduced density relative to wild-type alpha 2A and is not selectively uncoupled from a single signal transduction pathway, our findings of losses of alpha 2AAR-mediated functions in the D79N mice reflect a requirement for the alpha 2AAR subtype but do not reveal the importance of a specific signal transduction pathway. The alpha 2AAR subtype appears to mediate reduction in blood pressure following alpha 2A agonist administration as well as sedative, anesthetic-sparing, and analgesic responses to alpha 2AAR agonists. Therefore, the alpha 2AAR subtype appears to mediate a majority of the clinically relevant responses associated with alpha 2AAR agonist treatment.

Animals↗
PubMed1997

The alpha2a adrenergic receptor subtype mediates spinal analgesia evoked by alpha2 agonists and is necessary for spinal adrenergic-opioid synergy.

Agonists acting at alpha2 adrenergic and opioid receptors have analgesic properties and act synergistically when co-administered in the spinal cord; this synergy may also contribute to the potency and efficacy of spinally administered morphine. The lack of subtype-selective pharmacological agents has previously impeded the definition of the adrenergic receptor subtype(s) mediating these effects. We therefore exploited a genetically modified mouse line expressing a point mutation (D79N) in the alpha2a adrenergic receptor (alpha2aAR) to investigate the role of the alpha2aAR in alpha2 agonist-evoked analgesia and adrenergic-opioid synergy. In the tail-flick test, intrathecal administration of UK 14,304, a nonsubtype-selective alpha2AR agonist, had no analgesic effect in D79N mice, whereas the analgesic potency of morphine (intrathecal) in this assay was not affected by the mutation. The mutation also decreased alpha2-agonist-mediated spinal analgesia and blocked the synergy seen in wild-type mice with both the delta-opioid agonist deltorphin II and the micro-opioid agonist [D-ALA2,N-Me-Phe4, Gly-ol5]-Enkephalin (DAMGO) in the substance P behavioral test. In addition, the potency of spinally administered morphine was decreased in this test, suggesting that activation of descending noradrenergic systems impinging on the alpha2aAR contributes to morphine-induced spinal inhibition in this model. These results demonstrate that the alpha2aAR subtype is the primary mediator of alpha2 adrenergic spinal analgesia and is necessary for analgesic synergy with opioids. Thus, combination therapies targeting the alpha2aAR and opioid receptors may prove useful in maximizing the analgesic efficacy of opioids while decreasing total dose requirements.

Adrenergic alpha-Agonists↗
PubMed1997

Substitution of a mutant alpha2a-adrenergic receptor via "hit and run" gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo.

Norepinephrine contributes to antinociceptive, sedative, and sympatholytic responses in vivo, and alpha2 adrenergic receptor (alpha2AR) agonists are used clinically to mimic these effects. Lack of subtype-specific agonists has prevented elucidation of the role that each alpha2AR subtype (alpha2A, alpha2B, and alpha2C) plays in these central effects. Here we demonstrate that alpha2AR agonist-elicited sedative, anesthetic-sparing, and analgesic responses are lost in a mouse line expressing a subtly mutated alpha2AAR, D79N alpha2AAR, created by two-step homologous recombination. These functional changes are accompanied by failure of the D79N alpha2AAR to inhibit voltage-gated Ca2+ currents and spontaneous neuronal firing, a measure of K+ current activation. These results provide definitive evidence that the alpha2AAR subtype is the primary mediator of clinically important central actions of alpha2AR agonists and suggest that the D79N alpha2AAR mouse may serve as a model for exploring other possible alpha2AAR functions in vivo.

Analgesics↗
PubMed1997

Disruption of microtubules reveals two independent apical targeting mechanisms for G-protein-coupled receptors in polarized renal epithelial cells.

G-protein-coupled receptors demonstrate differing trafficking itineraries in polarized Madin-Darby canine kidney (MDCK II) cells. The alpha2A adrenergic receptor (alpha2AAR) is directly delivered to the basolateral subdomain; the A1 adenosine receptor (A1AdoR) is apically enriched in its targeting; and the alpha2BAR subtype is randomly delivered to both domains but selectively retained basolaterally (Keefer, J. R., and Limbird, L. E. (1993) J. Biol. Chem. 268, 11340-11347; Saunders, C., Keefer, J. R., Kennedy, A. P., Wells, J. N., and Limbird, L. E. (1996) J. Biol. Chem. 271, 995-1002; Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017-5024). The present studies explore the role of the polarized cytoskeleton in localization of G-protein-coupled receptors in MDCK II cells. Nocodazole or colchicine, which disrupt microtubules, did not perturb lateral localization of alpha2AR subtypes but led to a relocalization the A1AdoR to the basolateral surface, revealed by immunocytochemical and metabolic labeling strategies. Conversely, the apical component of the random delivery of alpha2BAR was not affected by these agents, suggesting microtubule-dependent and -independent apical targeting mechanisms for G-protein-coupled receptors in polarized cells. Apparent rerouting of the apically targeted A1AdoR was selective for microtubule-disrupting agents, since cytochalasin D, which disrupts actin polymerization, did not alter A1AdoR or alpha2BAR localization or targeting. These data suggest that multiple apical targeting mechanisms exist for G-protein-coupled receptors and that microtubule-disrupting agents serve as tools to probe their different trafficking mechanisms.

Animals↗
PubMed1997

Distribution of mRNA encoding three alpha 2-adrenergic receptor subtypes in the developing mouse embryo suggests a role for the alpha 2A subtype in apoptosis.

alpha 2-Adrenergic receptors (alpha 2-ARs) respond to norepinephrine and epinephrine to mediate diverse physiological effects. Using in situ hybridization, the expression pattern of the mRNA encoding the three alpha 2-AR subtypes (alpha 2A, alpha 2B, and alpha 2C) was examined in the mouse embryo. The mRNA encoding the three subtypes was first detected at stage 9.5 days postcoitus (d.p.c.) for the alpha 2A-AR (coincident with norepinephrine availability), 11.5 d.p.c. for the alpha 2B-AR, and 14.5 d.p.c. for the alpha 2C-AR subtype. The mRNA encoding the alpha 2A-AR subtype shows both the earliest and the most widespread expression pattern, including developing stomach and cecum, many craniofacial regions and areas in the central nervous system. Strikingly, the alpha 2A-AR mRNA is expressed in the interdigital mesenchyme between stage 12.5 and 14.5 d.p.c. in parallel with digit separation, raising the possibility that the alpha 2A-AR might contribute to the apoptotic events underlying this process. To test whether alpha 2A-AR can signal apoptotic events, the alpha 2A-AR subtype was introduced into two mouse mesenchymal cell lines, C3H/10t1/2 and NIH-3T3; expression of the alpha 2A-AR correlated with accelerated apoptosis, as detected both by the TUNEL assay and the loss of cell viability. In contrast to the wide distribution of mRNA encoding the alpha 2A-AR subtype, the alpha 2B-AR mRNA was detected only in the developing liver and was most readily detectable between 11.5 and 14.5 d.p.c., when the liver is the principal site of hematopoiesis. The alpha 2C-AR mRNA is detected in the nasal cavity and cerebellar primordium only at > or = 14.5 d.p.c. These studies represent the first characterization of the temporal and spatial expressions of the alpha 2A-AR, alpha 2B-AR, and alpha 2C-AR subtypes during embryogenesis and provide important insights concerning the loci and possible roles of alpha 2-AR-mediated regulation of physiological processes during the developmental program.

Animals↗
PubMed1996

Central hypotensive effects of the alpha2a-adrenergic receptor subtype.

alpha2-Adrenergic receptors (alpha2ARs) present in the brainstem decrease blood pressure and are targets for clinically effective antihypertensive drugs. The existence of three alpha2AR subtypes, the lack of subtype-specific ligands, and the cross-reactivity of alpha2AR agonists with imidazoline receptors has precluded an understanding of the role of individual alpha2AR subtypes in the hypotensive response. Gene targeting was used to introduce a point mutation into the alpha2aAR subtype in the mouse genome. The hypotensive response to alpha2AR agonists was lost in the mutant mice, demonstrating that the alpha2aAR subtype plays a principal role in this response.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1996

The three alpha 2-adrenergic receptor subtypes achieve basolateral localization in Madin-Darby canine kidney II cells via different targeting mechanisms.

The present studies examined the localization of the alpha2B- and alpha2C-adrenergic receptor (AR) subtypes in polarized Madin-Darby canine kidney cells (MDCK II) and the mechanisms by which this is achieved. Previously we demonstrated that the alpha2AAR subtype is directly delivered to lateral subdomain of MDCK II cells. Surface biotinylation strategies demonstrated that the alpha2BAR, like the alpha2AAR, achieves 85-90% basolateral localization at steady-state. However, in contrast to the alpha2AAR, this polarization occurs after initial random insertion of the alpha2BAR into both apical and basolateral surfaces followed by selective retention on the lateral subdomain (t1/2 the apical surface is 15-30 min; t1/2 the basolateral surface is 8-10 h). The alpha2CAR also is enriched on the basolateral surface at steady-state and, like the alpha2AAR, is directly delivered there. Morphological evaluation of the epitope-tagged alpha2AAR, alpha2BAR, and alpha2CAR subtypes by laser confocal microscopy not only corroborated the biochemically-defined basolateral localization of all three alpha2AR subtypes but also revealed that the alpha2CAR uniquely exists in an intracellular compartment(s) as well. Immunofluorescence due to intracellular alpha2CAR partially overlaps that due to calnexin, a marker for endoplasmic reticulum, as well as that due to mannosidase II, a marker for the trans-Golgi network. Taken together, the present findings demonstrate that the alpha2AAR, alpha2BAR, and alpha2CAR subtypes, which possess highly homologous structures and ultimately achieve similar polarization to the lateral surface of MDCK II cells, nonetheless manifest distinct trafficking itineraries.

Animals↗
PubMed1996

Receptors coupled to pertussis toxin-sensitive G-proteins traffic to opposite surfaces in Madin-Darby canine kidney cells. A1 adenosine receptors achieve apical and alpha 2A adrenergic receptors achieve basolateral localization.

The alpha 2A adrenergic receptor (alpha 2AAR) previously was shown to be directly delivered to and retained on the lateral subdomain of renal epithelial cells. The present studies demonstrate that, in contrast, wild-type and epitope-tagged canine A1 adenosine receptors (A1AdoR) are apically enriched (65-83%) in Madin-Darby canine kidney (MDCKII) and porcine renal epithelial (LLC-PKI) cells, based on surface biotinylation strategies detecting photoaffinity-labeled A1AdoR. Confocal microscopy corroborated the apical enrichment of the epitopetagged A1AdoR. Metabolic labeling studies revealed that this steady-state polarization is achieved by direct delivery to both the apical (60-75%) and basolateral surface. Growth of A1AdoR-expressing cells as monolayers presence of A1AdoR antagonists, which decreased cell growth, suggesting that A1AdoR elicit MDCKII cell proliferation. The preferential apical but detectable basolateral localization of A1AdoR provides a molecular understanding of published reports that functional responses can be elicited following apical as well as basolateral delivery of adenosine agonists in varying renal preparations. These findings also suggest that receptor chimeras derived from the Gi/Go-protein-coupled alpha 2AAR and A1AdoR will be informative in revealing structural features critical for basolateral versus apical targeting.

Amino Acid Sequence↗
PubMed1996

Expression of alpha 2-adrenergic receptor subtypes in the mouse brain: evaluation of spatial and temporal information imparted by 3 kb of 5' regulatory sequence for the alpha 2A AR-receptor gene in transgenic animals.

The present studies characterize the expression of the alpha 2A, alpha 2B and alpha 2C adrenergic receptor subtypes via in situ hybridization analysis of messenger RNA expression in the adult mouse brain, as well as the pattern of expression of alpha 2A adrenergic receptor messenger RNA at embryonic day E9.5, the earliest time for detection of the messenger RNA encoding this receptor subtype. alpha 2A adrenergic receptor messenger RNA is highly expressed in the sixth layer of the cortex and the locus coeruleus, alpha 2B adrenergic receptor messenger RNA predominantly in the thalamus and in the Purkinje layer of the cerebellum, and alpha 2C adrenergic receptor messenger RNA in the putamen caudate region of the mouse brain. Both alpha 2A and alpha 2C adrenergic receptor messenger RNA demonstrate strong expression in the amygdaloid complex, hypothalamus, olfactory system and the hippocampal formation. To develop a molecular understanding of the unique cellular expression of messenger RNA encoding the alpha 2A adrenergic receptor subtype, 2.83 kb of the upstream regulatory sequence for the alpha 2A adrenergic receptor gene was fused to the LacZ gene as a reporter gene and expression of beta-galactosidase activity was assessed in transgenic offspring. Although the spatial expression of the transgene in the adult brain often overlaps that for the endogenous alpha 2A adrenergic receptor, both ectopic expression and the absence of appropriate expression were noted; in contrast five of the six lines show temporal expression characteristic of the endogenous alpha 2A adrenergic receptor gene. The present studies provide the first characterization of messenger RNA localization for the three alpha 2 adrenergic receptor subtypes in the mouse CNS. Because the functional roles of the prazosin-sensitive alpha 2B adrenergic receptor and alpha 2C adrenergic receptor subtypes have been masked in most earlier physiological and pharmacological analyses of alpha 2 adrenergic receptor function, identifying the multiple loci alpha 2 adrenergic receptor subtype expression is an important prelude to understanding the functional roles of these three independent receptor populations in the mouse CNS. The findings in the transgenic animals indicating that approximately 3 kb of regulatory sequence has imparted faithful temporal but not spatial expression for the alpha 2A adrenergic receptor gene suggest that additional regulatory information is necessary for appropriate cell specific expression of messenger RNA for the alpha 2A adrenergic receptor subtype.

Amino Acid Sequence↗
PubMed1996

Genetic evidence for involvement of multiple effector systems in alpha 2A-adrenergic receptor inhibition of stimulus-secretion coupling.

The alpha 2A-adrenergic receptor (alpha 2AAR), via its interaction with the pertussis toxin-sensitive Gi/G(o) class of G proteins, modulates multiple effector systems, including inhibition of adenylyl cyclase and Ca2+ channels and activation of K+ channels. Mutation of a membrane-embedded aspartate residue, highly conserved among G protein-coupled receptors, in the alpha 2AAR to asparagine (D79N alpha 2AAR) results in selective uncoupling of the receptor to K+ currents but retention of inhibition of cAMP production and of voltage-sensitive Ca2+ currents when expressed in AtT20 anterior pituitary cells in culture. It is known that attenuation of cAMP synthesis alone cannot account for alpha 2AAR suppression of stimulus-secretion coupling; thus, the D79N alpha 2AAR provides a unique tool with which to assess the relative contribution of K+ current activation and Ca2+ current suppression in mediating the cellular responses of alpha 2AAR. The wild-type alpha 2AAR suppresses basal and secretagogue-evoked adrenocorticotropic hormone (ACTH) release in a manner indistinguishable from response to the endogenous somatostatin receptor. In contrast, the D79N alpha 2AAR does not attenuate basal ACTH release and is only partially effective in suppressing ACTH secretion evoked by the secretagogue isoproterenol. Regulation of ACTH release evoked by 8-bromo-cAMP, which bypasses receptor regulation of cAMP synthesis, suggests that attenuation of cAMP production, although not sufficient for inhibition of ACTH secretion, nevertheless participates in a functionally relevant manner. Taken together, the present findings indicate that alpha 2AAR-mediated suppression of neuropeptide secretion requires concomitant regulation of K+ and Ca2+ currents in parallel with attenuation of cAMP production.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Palmitoylation of the alpha 2A-adrenergic receptor. Analysis of the sequence requirements for and the dynamic properties of alpha 2A-adrenergic receptor palmitoylation.

Previous studies have demonstrated that the alpha 2A-adrenergic receptor (alpha 2A AR) incorporates [3H]palmitate and that replacement of Cys442 by Ala or Ser eliminates detectable acylation without perturbing coupling to pertussis toxin-sensitive GTP-binding proteins (Kennedy, M. E., and Limbird, L. E. (1993) J. Biol. Chem. 268, 8003-8011) or, as shown here, without perturbing agonist-dependent receptor phosphorylation, in contrast to the consequences of eliminating beta 2-adrenergic receptor acylation. As a first step in revealing the functional role for this post-translational modification at the alpha 2A AR, we explored sequences in the alpha 2AAR which confer alpha 2AAR acylation and whether or not [3H]palmitoylation of the alpha 2AAR is dynamic. Deletion of the 7 terminal amino acids distal to Cys442 of the alpha 2AAR did not eliminate detectable [3H]palmitoylation of the alpha 2AAR, whereas truncation to Leu441 did, indicating both that Cys442 is the likely site for acylation and that sequences distal to Cys442 are not required for acylation at Cys442. Since mutation of sequences proximal to Cys442 altered overall receptor structure, based on markedly reduced detectable adrenergic receptor binding, proximal motifs required for palmitoylation of the alpha 2AAR could not be explored further. When the turnover of [35S]Met/Cys-labeled alpha 2AAR was compared with the turnover of the [3H]palmitate-labeled alpha 2AAR, it was of interest that agonist treatment accelerated the half-life of decay of the [3H]palmitate-labeled alpha 2AAR without detectable receptor down-regulation, providing evidence that the acylation of the alpha 2AAR may be a dynamic process.

Acylation↗
PubMed1994

Mutation of an aspartate residue highly conserved among G-protein-coupled receptors results in nonreciprocal disruption of alpha 2-adrenergic receptor-G-protein interactions. A negative charge at amino acid residue 79 forecasts alpha 2A-adrenergic receptor sensitivity to allosteric modulation by monovalent cations and fully effective receptor/G-protein coupling.

We have mutated the aspartate residue in the putative second transmembrane spanning domain of the alpha 2A-adrenergic receptor (alpha 2AAR) to the non-negatively charged asparagine (D79N) and glutamine (D79Q) and the negatively charged glutamate (D79E) residue in an effort to better characterize the role of this residue, highly conserved among G-protein-coupled receptors, in Na+ regulation of ligand binding and in receptor G-protein coupling. Allosteric modulation of receptor-ligand interactions by Na+ is retained by the D79E alpha 2AAR but lost upon mutation to the uncharged D79N and D79Q residues. Loss of allosteric effects of Na+ is paralleled by a complete loss of retrograde information transfer from G-proteins to alpha 2AAR in AtT20 cells, measured via the sensitivity of radiolabeled agonist binding to Gpp(NH)p. In contrast to the complete elimination of retrograde signaling via the D79N and D79Q alpha 2AAR, anterograde information transfer from receptor to G-protein is modified in a more subtle quantitative way, since agonist-stimulated GTPase activity via D79N and D79Q alpha 2AAR, although apparently attenuated compared to wild type and D79E alpha 2AAR, is no less than the GTPase activity elicited by endogenous somatostatin receptors in AtT20 cells. These data indicate that a negative charge at amino acid residue 79 forecasts sensitivity to allosteric regulation by monovalent cations and its mutation to non-negatively charged residues elicits a nonparallel modulation of receptor-->G-protein versus G-protein-->receptor communication between alpha 2AAR and pertussis toxin-sensitive GTP-binding proteins.

Allosteric Regulation↗
PubMed1994

Unique structural features important for stabilization versus polarization of the alpha 2A-adrenergic receptor on the basolateral membrane of Madin-Darby canine kidney cells.

The alpha 2A-adrenergic receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.

Amino Acid Sequence↗