Interaction between GABA(A) receptor beta subunits and the multifunctional protein gC1q-R

gamma-Aminobutyric acid type A (GABA(A)) receptors were immunopurified from bovine brain using a monoclonal antibody directed against the alpha1 subunit. Of the several proteins that copurified, a 34-kDa protein was analyzed further. After enrichment and tryptic proteolysis, the resulting fragments were sequenced, and the protein was identified as gC1q-R. Using anti-gC1q-R and anti-GABA(A) receptor antibodies, mutual coimmunoprecipitation could be demonstrated from solubilized rat brain membranes. The stability of this interaction was estimated to be very high. Using the yeast two-hybrid system, various GABA(A) receptor subunit intracellular loop constructs were tested for an interaction with gC1q-R. All beta subunits, but not alpha 1 and gamma 2 subunits, were found to bind to gC1q-R. NH(2)- and COOH-terminally truncated beta 2 subunit loops were used to find the region responsible for the interaction with gC1q-R. A stretch of 15 amino acids containing 7 positively charged residues was identified (amino acids 399--413). This region contains residue Ser-410, which is a protein kinase substrate, and it is known that phosphorylation of this residue leads to an alteration in receptor activity. Localization studies suggested a predominantly intracellular localization. Our observations therefore suggest a tight interaction between gC1q-R and the GABA(A) receptor which might be involved in receptor biosynthesis or modulation of the mature function.     

Regulation of subcellular localization of alpha1-adrenoceptor subtypes

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the alpha1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with alpha1B-AR in vitro and in vivo through the specific site, and that in cells which co-express alpha1B-AR and gC1q-R, the subcellular localization of alpha1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of alpha1B-ARs.     

Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.     

gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes

InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.     

Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.     

Molecular cloning and expression of the cDNA for a novel alpha 1-adrenergic receptor subtype

A novel alpha 1-adrenergic receptor subtype has been cloned from a bovine brain cDNA library. The deduced amino acid sequence is that of a 466-residue polypeptide. The structure is similar to that of the other adrenergic receptors as well as the larger family of G protein-coupled receptors that have a presumed seven-membrane-spanning domain topography. The greatest sequence identity of this receptor protein is with the previously cloned hamster alpha 1B-adrenergic receptor being approximately 72% within the presumed membrane-spanning domains. Localization on different human chromosomes provides evidence that the bovine cDNA is distinct from the hamster alpha 1B-adrenergic receptor. The bovine cDNA clone expressed in COS7 cells revealed 10-fold higher affinity for the alpha 1-adrenergic antagonists WB4101 and phentolamine and the agonist oxymetazoline as compared with the alpha 1B receptor, results similar to pharmacologic binding properties described for the alpha 1A receptor. Despite these similarities in pharmacological profiles, the bovine alpha 1-adrenergic receptor is sensitive to inhibition by the alkylating agent chloroethylclonidine unlike the alpha 1A-adrenergic receptor subtype. In addition, a lack of expression in tissues where the alpha 1A subtype exists suggests that this receptor may actually represent a novel alpha 1-adrenergic receptor subtype not previously appreciated by pharmacological criteria.     

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.     

Characteristics for a salt-bridge switch mutation of the alpha(1b) adrenergic receptor. Altered pharmacology and rescue of constitutive activity

Agonist-dependent activation of the alpha(1)-adrenergic receptor is postulated to be initiated by disruption of an interhelical salt-bridge constraint between an aspartic acid (Asp-125) and a lysine residue (Lys-331) in transmembrane domains three and seven, respectively. Single point mutations that disrupt the charges of either of these residues results in constitutive activity. To validate this hypothesis, we used site-directed mutagenesis to switch the position of these amino acids to observe, if possible, regeneration of the salt-bridge reverses that the constitutive activity of the single point mutations. The transiently expressed switch mutant receptor displayed an altered pharmacological profile. The affinity of selective alpha(1b)-adrenergic receptor antagonists for the switch mutant (D125K/K331D) was no different from the wild-type alpha(1b)-adrenergic receptor, suggesting that both receptors are maintaining similar tertiary structures in the cell membrane. However, there was a significant 4-6-fold decrease in the affinity of protonated amine receptor agonists and a 3-6-fold increase in the affinity of carboxylated catechol derivatives for the switch mutant compared with the wild-type alpha(1b)-adrenergic receptor. This pharmacology is consistent with a reversed charge at position 125 in transmembrane domain three. Interestingly, the ability of either a negatively or positively charged agonist to generate soluble inositol phosphates was similar for both types of receptors. Finally, the switch mutant (D125K/K331D) displayed similar basal signaling activity as the wild-type receptor, reversing the constitutive activity of the single point mutations (D125K and K331D). This suggests an ionic constraint has been reformed in the switch mutant analogous to the restraint previously described for the wild-type alpha(1b)-adrenergic receptor. These results strongly establish the disruption of an electrostatic interaction as an initial step in the agonist-dependent activation of alpha(1)-adrenergic receptors.     

Molecular cloning and initial characterization of African green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells

We report the expression of endogenous CRF1 in COS-7 cells (African green monkey origin). Cloning of the coding region of CRF1 gene identified three alternatively spliced isoforms with nucleotide and predicted amino acid sequences corresponding to the membrane bound alpha and c and soluble e isoforms. DNA sequencing of the main isoform CRF1alpha showed homologies of 99%, 97% and 91% with the rhesus monkey, human and rodent genes, respectively; the deduced protein sequence differed in only one amino acid with rhesus monkey and human. Western blot analysis with antibodies against human CRF1 demonstrated immunoreactive proteins with MW of 37, 52, 70 and 80-85 in crude membrane or cytoplasm preparation; two additional species of 40 and 60 kDa were detected only in the cytoplasmic fraction. On immunocytochemistry CRF1 was localized to both the cell surface and intracellularly. The receptor was functional, e.g., addition of CRF to COS-7 cells inhibited cell proliferation and stimulated release of arachidonic acid; nevertheless, it was poorly coupled to cAMP production (its stimulation was minimal in native cells). In conclusion, COS cells that are routinely used for the study of transfected CRF receptors do express endogenous CRF1 mRNA with splicing behavior similar to that reported in human and rodent cells, and translated into functional CRF1 receptors.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.     

Interaction of the amyloid precursor like protein 1 with the alpha2A-adrenergic receptor increases agonist-mediated inhibition of adenylate cyclase

Alpha2-adrenergic receptor agonists exert potent analgesic and sedative/hypnotic effects. In addition, they have been shown to be neuroprotective, but the mechanisms of these actions are still poorly defined. To isolate proteins that may control alpha2-adrenergic receptor function or trafficking, we performed a two-hybrid screen using the carboxy-terminal fourth intracellular tail of the alpha2A-adrenergic receptor as bait. This screen identified the amyloid precursor like protein 1 (APLP1), a homologue of the beta-amyloid precursor protein involved in Alzheimer's disease, as alpha2A-adrenergic receptor-binding protein. GST affinity chromatography revealed that APLP1 specifically interacts with all three human alpha2-adrenergic receptor subtypes and deletion mutant analysis confined the APLP1 domain involved in binding to alpha2-adrenergic receptors to the 13 amino acid residues Ser599-Ala611. Coimmunoprecipitations of transiently transfected cells with epitope-tagged APLP1 and alpha2-adrenergic receptors confirmed the interaction. Agonist treatment tended to increase the amount of alpha2A-adrenergic receptor associated with APLP1 while coimmunoprecipitations were not affected by the state of receptor phosphorylation or cotransfection of arrestin-3. Confocal laser microscopy showed that APLP1 causes a considerable shift of the alpha2A-adrenergic receptor localization from plasma membrane to intracellular compartments. Furthermore, cotransfection of alpha2A-adrenergic receptor and APLP1 into HEK293 cells significantly increased norepinephrine mediated inhibition of adenylate cyclase activity. These results suggest a possible role of APLP1 in regulation of alpha2A-adrenergic receptor trafficking. Moreover, we speculate that this interaction may present one mechanism by which alpha2-adrenergic receptor agonists exert their neuroprotective effects.     

Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

Cloning, sequence analysis, and permanent expression of a human alpha 2-adrenergic receptor in Chinese hamster ovary cells. Evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation

The gene encoding a human alpha 2-adrenergic receptor was isolated from a human genomic DNA library using a 367-base pair fragment of Drosophila genomic DNA that exhibited 54% identity with the human beta 2-adrenergic receptor and 57% identity with the human alpha 2-adrenergic receptor. The nucleotide sequence of a fragment containing the human alpha 2-receptor gene and 2.076 kilobases of untranslated 5' sequence was determined, and potential upstream regulatory regions were identified. This gene encodes a protein of 450 amino acids and was identified as an alpha 2-adrenergic receptor by homology with published sequences and by pharmacological characterization of the protein expressed in cultured cells. Permanent expression of the alpha 2-receptor was achieved by transfecting Chinese hamster ovary (CHO) cells which lack adrenergic receptors with a 1.5-kilobase NcoI-HindIII fragment of the genomic clone containing the coding region of the gene. The alpha 2-receptor expressed in CHO cells displayed pharmacology characteristic of an alpha 2 A-receptor subtype with a high affinity for yohimbine (Ki = 1 nM) and a low affinity for prazosin (Ki = 10,000 nM). Agonists displayed a rank order of potency in radioligand binding assays of para-aminoclonidine greater than or equal to UK-14304 greater than (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol, consistent with the identification of this protein as an alpha 2-receptor. The role of the alpha 2-receptor in modulating intracellular cyclic AMP concentrations was investigated in three transfected cell lines expressing 50, 200, and 1200 fmol of receptor/mg membrane protein. At low concentrations (1-100 nM), (-)-epinephrine attenuated forskolin-stimulated cyclic AMP accumulation by up to 60% in a receptor density-dependent manner. At epinephrine concentrations above 100 nM, cyclic AMP levels were increased up to 140% of the forskolin-stimulated level. Pertussis toxin pretreatment of cells eliminated alpha 2-receptor-mediated attenuation of forskolin-stimulated cyclic AMP levels and enhanced the receptor density-dependent potentiation of forskolin-stimulated cyclic AMP concentrations from 3 to 8-fold. Potentiation of forskolin-stimulated cyclic AMP levels was also elicited by the alpha 2-adrenergic agonists, UK-14304 and para-aminoclonidine, and blocked by the alpha 2-adrenergic antagonist yohimbine, but not by the alpha 1-adrenergic antagonist prazosin or the beta-adrenergic antagonist propranolol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cooperation of C1q receptors and integrins in C1q-mediated endothelial cell adhesion and spreading

The interaction of C1q with endothelial cells elicits a multiplicity of biologic responses. Although these responses are presumed to be mediated by the interaction of C1q with endothelial cell surface proteins, the identity of the participants is not known. In this study we examined the roles of two C1q binding proteins, cC1q-R/calreticulin and gC1q-R/p33, in C1q-mediated adhesion and spreading of human dermal microvascular endothelial cells (HDMVEC). When HDMVEC were cultured in microtiter plate wells coated with concentrations of C1q ranging from 0 to 50 microg/ml, a specific and dose-dependent adhesion and spreading was observed. The extent of adhesion and spreading was similar to the adhesion seen on collagen-coated wells. Spreading (68 +/- 12%) and to a moderate extent adhesion (47 +/- 9%) were inhibited by anti-gC1q-R mAb 60.11. Similar effects were noted with polyclonal anti-cC1q-R but not with control nonimmune IgG. The two Abs had a slight additive effect (75 +/- 13% inhibition) when mixed together in the proportion of 100 microg/ml anti-gC1q-R and 30 microg/ml anti-cC1q-R. More importantly, a 100% inhibition of spreading, but not adhesion, to C1q-coated wells was observed when HDMVEC were cultured in the presence of 30 microM of the peptide GRRGDSP but not GRRGESP. Furthermore, while anti-beta(1) integrin Ab blocked both adhesion and spreading, anti-alpha(5) integrin blocked only spreading and not adhesion. Ag capture ELISA of endothelial cell membrane proteins using polyclonal anti-gC1q-R showed the presence of not only beta(1) and alpha(5) integrins but also CD44. Taken together these results suggest that endothelial cell adhesion and spreading require the cooperation of both C1qRs and beta(1) integrins and possibly other membrane-spanning molecules.     

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)     

The N-terminal extracellular domain 23-60 of the calcitonin receptor-like receptor in chimeras with the parathyroid hormone receptor mediates association with receptor activity-modifying protein 1

The calcitonin receptor-like receptor (CLR) requires the associated receptor activity-modifying protein (RAMP)1 to reveal a calcitonin gene-related peptide (CGRP) receptor. Here, the subdomain of the CLR that associates with RAMP1 has been identified in chimeras between the CLR and the parathyroid hormone (PTH) receptor 1 (PTHR). The PTHR alone does not interact with RAMP1. RAMP1 requires the CLR for its transport to the cell surface. Thus, receptor-dependent RAMP1 delivery to the plasma membrane and coimmunoprecipitation from the cell surface were used as measures for receptor/RAMP1 interaction. Several chimeric CLR-PTHR included the N-terminal amino acids 23-60 of the CLR transported RAMP1 to the surface of COS-7 cells much like the intact CLR. Moreover, RAMP1 coimmunoprecipitated with these receptors from the cell surface. A CLR deletion mutant, consisting of the N-terminal extracellular domain, the first transmembrane domain, and the C-terminal intracellular region, revealed the same results. Cyclic AMP was stimulated by CGRP in CLR/RAMP1 expressing cells (58 +/- 19-fold, EC(50) = 0.12 +/- 0.03 nM) and by PTH-related protein in cells expressing the PTHR (50 +/- 10-fold, EC(50) = 0.25 +/- 0.03 nM) or a PTHR with the N-terminal amino acids 23-60 of the CLR (23 +/- 5-fold, EC(50) > 1000 nM). Other chimeric CLR-PTHR were inactive. In conclusion, structural elements in the extreme N-terminus of the CLR between amino acids 23-60 are required and sufficient for CLR/RAMP1 cotransport to the plasma membrane and heterodimerization.     

Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. [3H]Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, [3H]Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.