Agonist-specific desensitization of PGE2-stimulated cAMP signaling due to upregulated phosphodiesterase expression in human lung fibroblasts.

Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3',5'-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.

Leu8 and Pro8 oxytocin agonism differs across human, macaque, and marmoset vasopressin 1a receptors.

Oxytocin (OXT) is an important neuromodulator of social behaviors via activation of both oxytocin receptors (OXTR) and vasopressin (AVP) 1a receptors (AVPR1a). Marmosets are neotropical primates with a modified OXT ligand (Pro8-OXT), and this ligand shows significant coevolution with traits including social monogamy and litter size. Pro8-OXT produces more potent and efficacious responses at primate OXTR and stronger behavioral effects than the consensus mammalian OXT ligand (Leu8-OXT). Here, we tested whether OXT/AVP ligands show differential levels of crosstalk at primate AVPR1a. We measured binding affinities and Ca2+ signaling responses of AVP, Pro8-OXT and Leu8-OXT at human, macaque, and marmoset AVPR1a. We found that AVP binds with higher affinity than OXT across AVPR1a, and marmoset AVPR1a show a 10-fold lower OXT binding affinity compared to human and macaque AVPR1a. Both Leu8-OXT and Pro8-OXT produce a less efficacious response than AVP at human AVPR1a and higher efficacious response than AVP at marmoset AVPR1a. These data suggest that OXT might partially antagonize endogenous human AVPR1a signaling and enhance marmoset AVPR1a signaling. These findings aid in further understanding inconsistencies observed following systemic intranasal administration of OXT and provide important insights into taxon-specific differences in nonapeptide ligand/receptor coevolution and behavior.

Binding Characteristics of Two Oxytocin Variants and Vasopressin at Oxytocin Receptors from Four Primate Species with Different Social Behavior Patterns.

A clade of New World monkeys (NWMs) exhibits considerable diversity in both oxytocin (OT) ligand and oxytocin receptor (OTR) structure. Most notable is the variant Pro8-OT, with proline instead of leucine at the eighth position, resulting in a rigid bend in the peptide backbone. A higher proportion of species that express Pro8-OT also engage in biparental care and social monogamy. When marmosets (genus Callithrix), a biparental and monogamous Pro8-OT NWM species, are administered the ancestral Leu8-OT, there is no change in social behavior compared with saline treatment. However, when Pro8-OT is administered, marmosets' sociosexual and prosocial behaviors are altered. The studies here tested the hypothesis that OTR binding affinities and OT-induced intracellular Ca2+ potencies would favor the native OT ligand in OTRs from four primate species, each representing a unique combination of ancestral lineage, breeding system, and native OT ligand: humans (Leu8-OT, monogamous, apes), macaques (Leu8-OT, nonmonogamous, Old World monkey), marmosets (Pro8-OT, monogamous, NWM), and titi monkeys (Leu8-OT, monogamous, NWM). OTRs were expressed in immortalized Chinese hamster ovary cells and tested for intact-cell binding affinities for Pro8-OT, Leu8-OT, and arginine vasopressin (AVP), as well as intracellular Ca2+ signaling after stimulation with Pro8-OT, Leu8-OT, and AVP. Contrary to our hypothesis, Pro8-OT bound at modestly higher affinities and stimulated calcium signaling at modestly higher potencies compared with Leu8-OT in all four primate OTRs. Thus, differences downstream from a ligand-receptor binding event are more likely to explain the different behavioral responses to these two ligands.

Biphasic changes in airway epithelial cell EGF receptor binding and phosphorylation induced by components of hogbarn dust.

PURPOSE OF THE STUDY: Workers in enclosed hogbarns experience an increased incidence of airway inflammation and obstructive lung disease, and an aqueous hogbarn dust extract (HDE) induces multiple inflammation-related responses in cultured airway epithelial cells. Epidermal growth factor receptor (EGFR) phosphorylation and activation has been identified as one important mediator of inflammatory cytokine release from these cells. The studies here investigated both early and late phase adaptive changes in EGFR binding properties and subcellular localization induced by exposure of cells to HDE. MATERIALS AND METHODS: Cell surface EGFRs were quantified as binding to intact cells on ice. EGFR phosphorylation, expression, and localization were assessed with anti-EGFR antibodies and either blotting or confocal microscopy. RESULTS: In BEAS-2B and primary human bronchial epithelial cells, HDE induced decreases in cell surface EGFR binding following both 15-min and 18-h exposures. In contrast, H292 cells exhibited only the 15-min decrease, with binding near the control level at 18 hr. Confocal microscopy showed that the 15-min decrease in binding is due to EGFR endocytosis. Although total EGFR immunoreactivity decreased markedly at 18 hr in confocal microscopy with BEAS-2B cells, immunoblots showed no loss of EGFR protein. HDE stimulated EGFR phosphorylation at both 15 min and 18 hr in BEAS-2B cells and primary cells, but only at 15 min in H292 cells, indicating that the different EGFR binding changes among these cell types is likely related to their different time-dependent changes in phosphorylation. CONCLUSIONS: These studies extend the evidence for EGFRs as important cellular targets for components of HDE and they reveal novel patterns of EGFR phosphorylation and binding changes that vary among airway epithelial cell types. The results provide both impetus and convenient assays for identifying the EGFR-activating components and pathways that likely contribute to hogbarn dust-induced lung disease in agricultural workers.

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis.

Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of (125)I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl-CIN85-endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.

Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation.

BACKGROUND: The balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. This study was designed to investigate the effect of budesonide on fibroblast-mediated tissue repair and remodeling. METHODS: Using human fetal lung fibroblasts in a three-dimensional collagen gel culture system, we investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of Inflammatory cytokines (interleukin-1ß and tumor necrosis factor α; 5 ng/mL each) and, in order to activate latent proteases, serine protease trypsin 0.25 µg/mL. The effects of budesonide on metalloproteinase production and activation were also investigated. RESULTS: Inflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, P < 0.01). Budesonide 100 nM almost completely inhibited the release and mRNA expression of metalloproteinase-1, metalloproteinase-3, and metalloproteinase-9 induced by the cytokines (P< 0.05). Exposure to the cytokines plus trypsin increased collagen degradation and conversion of the metalloproteinases to lower molecular weight forms corresponding to their active forms. Budesonide blocked both enhanced collagen degradation (P< 0.01) and suppressed trypsin-mediated conversion of cytokine-induced metalloproteinase-9 and metalloproteinase-3 to lower molecular weight forms. Similar effects were observed with dexamethasone 1 µM, suggesting a class effect. CONCLUSION: These findings demonstrate that budesonide directly modulates contraction of collagen gels and can decrease collagen degradation under Inflammatory conditions. The mechanism of this effect is through suppressing gene expression, release, and activation of metalloproteinases. By modulating the release and activity of metalloproteinases, inhaled budesonide may be able to modify airway tissue repair and remodeling.

Development of a phosphatase-resistant, L-tyrosine derived LPA1/LPA3 dual antagonist.

Lysophosphatidic acid (LPA) is a bioactive compound that has gained attention due to its role in neoplastic diseases. Our group has developed a potent dual LPA1/LPA3 receptor antagonist, VPC51098 (LPA1 IC(50) = 84 nM, LPA1 IC(50) = 48 nM) that contained a labile phosphate head group. This lability has impaired our evaluation of our scaffold of LPA receptor antagonists in vivo. We wished to replace the phosphate with a potentially more stable head group while retaining potency at both LPA1 and LPA3 to facilitate future in vivo studies. We tested in vitro potency of all head groups including α-methylene, α-fluoromethylene, α-hydroxymethylene; vinyl phosphonates; α-fluoro vinyl phosphonates. The most potent compound was found to be a low micromolar inhibitor VPC51299 that contained a vinyl phosphonate and possessed a half-life of approximately 90 min in rats when dosed intravenously. Herein, we describe the synthesis and initial biological evaluation of these compounds.

Airway epithelial epidermal growth factor receptor mediates hogbarn dust-induced cytokine release but not Ca2+ response.

A subset of workers in swine confinement facilities develops chronic respiratory disease. An aqueous extract of dust from these facilities (hogbarn dust extract [HDE]) induces IL-6 and IL-8 release and several other responses in isolated airway epithelial cells. The cell membrane receptors by which HDE initiates these responses have not been identified. Because several other inhaled agents induce airway epithelial cell responses through epidermal growth factor receptor (EGFR) activation, we hypothesized that HDE would activate EGFRs and that EGFRs would be required for some of the responses to HDE. Exposure of Beas-2B cells to HDE caused EGFR phosphorylation and downstream ERK activation, and both responses were blocked by the EGFR-selective kinase inhibitor AG1478. AG1478 and EGFR-neutralizing antibody reduced HDE-stimulated IL-6 and IL-8 release by about half. Similar EGFR phosphorylation and requirement of EGFRs for maximal IL-6 and IL-8 release were found with primary isolates of human bronchial epithelial cells. Because HDE-stimulated IL-6 and IL-8 release involve the Ca(2+)-dependent protein kinase Cα, we hypothesized that HDE would induce intracellular Ca(2+) mobilization. HDE exposure induced intracellular Ca(2+) mobilization in Beas-2B cells and in primary cell isolates, but this response was neither mimicked by EGF nor inhibited by AG1478. Thus, HDE activates EGFRs and their downstream signaling, and EGFR activation is required for some but not all airway epithelial cell responses to HDE.

Modulation of epidermal growth factor receptor binding to human airway smooth muscle cells by glucocorticoids and beta2-adrenergic receptor agonists.

EGF receptors (EGFRs) are increased in airway smooth muscle in asthma, which may contribute to both their hyperproliferation and hypercontractility. Lysophosphatidic acid (LPA) is a candidate pathological agent in asthma and other airway diseases, and LPA upregulates EGFRs in human airway smooth muscle (HASM) cells. We tested whether therapeutic glucocorticoids and/or beta(2)-adrenergic receptor (beta(2)AR) agonists also alter EGFR binding in HASM cells. Exposure to glucocorticoids for 24 h induced a twofold increase in EGFR binding similar to that with LPA; fluticasone was markedly more potent than dexamethasone. The increase in EGFR binding by glucocorticoids required 24-h exposure, consistent with transcription-mediated effects. Although the increase in EGFR binding was blocked by the protein synthesis inhibitor cycloheximide for LPA, fluticasone, and dexamethasone, only LPA induced a significant increase in EGFR protein expression detected by immunoblotting. In contrast to the increased binding induced by the glucocorticoids, the beta(2)AR agonists isoproterenol, albuterol, and salmeterol all induced a decrease in EGFR binding. beta(2)AR agonist effects were multiphasic, with an initial decline at 2-4 h that reversed by 6 h and a second, somewhat greater decrease by 18-24 h. In cells pretreated with glucocorticoids, the decreases in EGFR binding by subsequent beta(2)AR treatment were not statistically significant; glucocorticoid upregulation of EGFRs also prevented further increases by LPA. Similar increases by glucocorticoids and decreases by beta(2)AR agonists were found in HFL-1 human lung fibroblasts. These complex and opposing effects of clinically relevant glucocorticoids and beta(2)AR agonists on airway mesenchymal cell EGFRs likely contribute to their overall therapeutic profile in the diseased airway.

Lysophosphatidic acid induces rapid and sustained decreases in epidermal growth factor receptor binding via different signaling pathways in BEAS-2B airway epithelial cells.

Lysophosphatidic acid (LPA) and epidermal growth factor (EGF) are important mediators of lung cell function and lung diseases. We showed previously that LPA decreases epidermal growth factor receptor (EGFR) binding rapidly in BEAS-2B airway epithelial cells, and this decrease is sustained to at least 18 h. The current studies investigate which LPA signaling pathways mediate the rapid versus sustained decreases in EGFR binding in BEAS-2B cells. The G(i/o) inhibitor pertussis toxin and the Rho kinase inhibitor Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] had no effect on the rapid or sustained decreases. However, the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)-butadiene ethanolate] decreased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, completely inhibited the rapid decrease in binding, and partially inhibited the sustained decrease. The direct Ca2+- and phospholipid-dependent protein kinase (PKC) activator phorbol-12-myristate-13-acetate stimulated ERK1/2 phosphorylation and decreased EGFR binding at both 15 min and 18 h. Furthermore, inhibitors of PKC partially inhibited ERK1/2 phosphorylation and the 15-min decrease but completely inhibited the 18-h decrease. Inhibitor time course studies showed that PKC induction of the 18-h decrease occurred during the first 3 h of treatment. We showed previously that LPA-stimulated EGFR transactivation contributes to the rapid decrease. Two transactivation inhibitors partially inhibited ERK1/2 phosphorylation, and U0126 partially inhibited EGFR transactivation, indicating that MEK may be involved both upstream and downstream of EGFR activation. Together, the data presented here indicate that LPA mediates the rapid decrease in EGFR binding via EGFR transactivation, MEK/ERK, and PKC, whereas the sustained decrease is regulated primarily by PKC.

Lysophosphatidic acid decreases epidermal growth factor receptor binding in airway epithelial cells.

We showed previously that treatment of human airway smooth muscle cells and lung fibroblasts with lysophosphatidic acid (LPA) increases the binding of epidermal growth factor (EGF) to EGF receptors (EGFRs). The purpose of this study was to determine whether LPA also regulates EGFR binding in airway epithelial cells. Airway epithelial cells were incubated in the absence or presence of 10 microM LPA for increasing times, and binding of 125I-EGF to intact cells on ice was measured. Exposure to LPA for only 15 min caused a 30 to 70% decrease in EGFR binding in a dose-dependent manner, depending on the cell line. This decrease in binding was sustained to at least 18 h in BEAS-2B and primary human bronchial epithelial cells. In contrast, the LPA-induced decrease in binding reversed rapidly in two lung cancer epithelial cell lines, H292 and A549, returning to control levels within 3 h. LPA increased phosphorylation of the EGFR in BEAS-2B cells, and this phosphorylation was inhibited by both 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478; EGFR tyrosine kinase inhibitor) and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001; matrix metalloproteinase inhibitor) but not by CRM197 (heparin-binding EGF inhibitor). AG-1478 and GM6001 also inhibited the LPA-induced decrease in EGFR binding but only by 50%, suggesting only partial involvement of EGFR transactivation in the decrease in EGFR binding. In summary, LPA stimulates a decrease in EGFR binding in airway epithelial cells that is sustained in normal cells but that rapidly reverses in cancer cells. LPA-induced transactivation of EGFRs occurs and contributes to the decrease in EGFR binding, but additional pathway(s) may also be involved.

The N terminus of the human alpha1D-adrenergic receptor prevents cell surface expression.

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A)NT-D, alpha(1B)NT-D, alpha(1D)NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)-or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

Regulation of alpha-1B adrenergic receptor localization, trafficking, function, and stability.

The alpha-1 adrenergic receptors (alpha(1)ARs) play important roles in normal physiology and in many disease states, and understanding their signaling pathways and regulatory mechanisms is thus of considerable relevance, in particular for identifying pharmacological targets for therapeutic modulation. The expression, function, localization, trafficking, and stability of these receptors are all subject to complex regulation by diverse molecular mechanisms. This article highlights recent studies from our laboratory and others focused on the localization and trafficking of the alpha-1B adrenergic receptor (alpha(1B)AR) subtype and on changes in its stability that are likely to be involved in regulating receptor expression. The role(s) of protein kinase C in alpha(1B)AR sequestration, endocytosis, and extracellular signal-regulated kinase (ERK) activation are summarized, and evidence for alpha(1B)AR localization in caveolae/rafts is presented. Receptor structural domains involved in the multiple steps and mechanisms of agonist-induced desensitization are described. Finally, aspects of alpha(1B)AR structural stability that appear to control its drug-induced up- and down-regulation are discussed. Our understanding of regulation for the alpha(1B)AR subtype provides a model for studies of the differential regulation of the other alpha(1)AR subtypes and may lead to identification of new molecular targets for therapeutic intervention in a variety of disease states.

Up-regulation of alpha1B-adrenergic receptors with defects in G protein coupling: ligand-induced protection from receptor instability.

The biochemical basis for the unexpected agonist-induced up-regulation of the number of radioligand binding sites for two mutated alpha1B-adrenergic receptors reported previously was investigated. Up-regulation was independent of the expression vector used and was not prevented by cycloheximide or actinomycin D, eliminating several potential transcriptional mechanisms and new receptor protein synthesis. Antagonists were also able to induce up-regulation, suggesting that ligand occupancy without signal generation was sufficient to induce the increase in binding sites. Accordingly, we hypothesized that up-regulation results from ligand-induced protection from inherent instability of these mutated receptors. Studies with receptors in isolated membranes revealed that the two mutated receptors that exhibited up-regulation in intact cells also exhibited an inherent instability of their ligand binding capacity, and binding of either agonists or antagonists to these receptors could protect against the loss of binding. In contrast, the wild-type receptor and other mutated receptors that did not exhibit up-regulation in intact cells did not exhibit instability or ligand-induced protection in isolated membranes. The occurrence of instability and protection in isolated membranes for only those mutated receptors and ligands that exhibit up-regulation in intact cells provides compelling evidence that the apparent up-regulation of binding sites in intact cells results from ligand-induced protection from an inherent instability of these G protein coupling-defective receptors. Inclusion of protease inhibitors markedly reduced the loss of binding in isolated membranes, implicating membrane-localized proteolysis as the likely mechanism for the instability.

Regulatory properties of alpha(1B)-adrenergic receptors defective in coupling to phosphoinositide hydrolysis.

Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for alpha(1B)-adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and down-regulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The Delta12 and Delta5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted (Delta[246-261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by alpha(1B)-adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes.