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alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR >> alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

The I(2) subgroup of imidazoline-binding sites was identified as monoamine oxidases (MAOs), but it is unclear whether there are I(2)-binding sites located on proteins distinct from MAOs. To address this issue, we characterized I(2)-binding proteins in liver and brain of wild-type and MAO A- and MAO B-deficient mice. I(2)-binding sites were identified using [(3)H]idazoxan and the photoaffinity adduct 2-[3-azido-4-[(125)I]iodophenoxyl]methylimidazoline ([(125)I]AZIPI). [(3)H]Idazoxan labeled binding sites with ligand recognition properties typical of I(2) sites in both brain and liver of wild-type mice. High-affinity, specific [(3)H]idazoxan binding were not altered in MAO A knockout (KO) mice. In contrast, [(3)H]idazoxan binding was completely abolished in both liver and brain of MAO B KO mice. In wild-type mice, [(125)I]AZIPI photolabeled three proteins with apparent molecular masses of approximately 28 (liver), approximately 61 (brain), and approximately 55 kDa (liver and brain). The photolabeling of each protein was blocked by the imidazoline cirazoline (10 microM). Photolabeling of the approximately 61- and approximately 55-kDa proteins was not observed in MAO A and B KO mice, respectively. In contrast, photolabeling of the liver approximately 28-kDa protein was still observed in MAO-deficient mice, indicating that this protein is unrelated to MAOs. These data indicate that I(2) imidazoline-binding sites identified by [(3)H]idazoxan reside solely on MAO B. The binding sites on MAO A and the liver approximately 28-kDa protein may represent additional subtypes of the family of the imidazoline-binding sites.

Gbetagamma binds directly to the third intracellular (i3) loop subdomain of the M(3)-muscarinic receptor (MR). In this report, we identified the Gbetagamma binding motif and G-protein-coupled receptor kinase (GRK2) phosphorylation sites in the M(3)-MR i3 loop via a strategy of deletional and site-directed mutagenesis. The Gbetagamma binding domain was localized to Cys(289)-His(330) within the M(3)-MR-Arg(252)-Gln(490) i3 loop, and the binding properties (affinity, influence of ionic strength) of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain were similar to those observed for the entire i3 loop. Site-directed mutagenesis of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain indicated that Phe(312), Phe(314), and a negatively charged region (Glu(324)-Asp(329)) were required for interaction with Gbetagamma. Generation of the full-length M(3)-MR-Arg(252)-Gln(490) i3 peptides containing the F312A mutation were also deficient in Gbetagamma binding and exhibited a reduced capacity for phosphorylation by GRK2. A similar, parallel strategy resulted in identification of major residues ((331)SSS(333) and (348)SASS(351)) phosphorylated by GRK2, which were just downstream of the Gbetagamma binding motif. Full-length M(3)-MR constructs lacking the 42-amino acid Gbetagamma binding domain (Cys(289)-His(330)) or containing the F312A mutation exhibited ligand recognition properties similar to wild type receptor and also effectively mediated agonist-induced increases in intracellular calcium following receptor expression in Chinese hamster ovary and/or COS 7 cells. However, the M(3)-MRDeltaCys(289)-His(330) and M(3)-MR(F312A) constructs were deficient in agonist-induced sequestration, indicating a key role for the Gbetagamma-M(3)-MR i3 loop interaction in receptor regulation and signal processing.

A series of phenoxy-substituted methylimidazoline derivatives were synthesized and used to define the ligand recognition properties of the imidazoline-binding domain (IBD) on monoamine oxidase (MAO)-B and its role in substrate processing. The rank order of potency for selected compounds in competitive binding studies with the imidazoline [(3)H]idazoxan was different from that in enzyme activity assays, suggesting that the IBD and the site involved in enzyme inhibition are distinct. IC(50) values for inhibition of MAO-B activity by imidazoline/guanidinium ligands were one to two orders of magnitude greater than ligand concentrations that probably saturate the IBD, but were equal to the K(d) values of these ligands in competitive binding assays with the reversible MAO-B inhibitor [(3)H]Ro 19-6327. In addition, the degree of enzyme inhibition by these ligands was similar in platelet and liver, tissues exhibiting 10-fold differences in the amount of the IBD-accessible enzyme subpopulation. These data suggested that the inhibitory effect of these compounds on MAO-B activity involved a secondary interaction with the enzyme domain recognizing the inhibitor Ro 19-6327 and does not involve interaction with the IBD. Subsequent radioligand-binding studies indicated that human liver MAO-B actually existed as two distinct populations that differed in the accessibility of their IBD. The relatively small amounts of MAO-B possessing an accessible IBD ( approximately 5% in human liver) precludes determination of the functional consequences of ligand binding to the IBD. This subpopulation of MAO-B may be selectively regulated or generated in different individuals or tissues and targeted by pharmacologically active compounds in a cell type-specific manner.

An agonist at a specific G protein-coupled receptor may exhibit a range of efficacies for any given response in a cell-specific manner. We report that the relationship between different states of agonism is regulated by the type of G protein expressed in the cell. In NIH-3T3 alpha(2)-adrenergic receptor (AR) transfectants, the alpha(2)-AR agonists clonidine, oxymetazoline, UK-14304, and epinephrine increased [(35)S]guanosine-5'-O-(3-thio)triphosphate binding in a dose-dependent manner from a basal value of 101.2 +/- 6. 5 fmol/mg to a maximal response (100 microM) of 196.6 +/- 9.8, 182.3 +/- 2, 328.1 +/- 11.2, and 340.6 +/- 3 fmol/mg, respectively. Thus, clonidine and oxymetazoline behaved as partial agonists. Receptor-mediated activation of G proteins in membrane preparations was blocked by cell pretreatment with pertussis toxin, indicating receptor coupling to the subgroup of pertussis toxin-sensitive G protein (Gialpha2,3) expressed in NIH-3T3 cells. Ectopic expression of Goalpha1 but not Gialpha1 increased the relative efficacy of clonidine and oxymetazoline such that the two ligands now behaved as close to full agonists in this assay system. The relationship between full and partial agonists in the different genetic backgrounds was not altered by progressive reduction in the amount of G protein available for coupling to receptor. The increased efficacy observed for clonidine in the Goalpha1 transfectants was not due to changes in the relative affinities or amounts of high-affinity, Gpp(NH)p-sensitive binding of agonist. These data suggest that there is little difference in the ability of clonidine to interact with or stabilize alpha(2)-AR-Gialpha2/Gialpha3 versus alpha(2)-AR-Goalpha1 complexes, but that the subsequent step of signal transfer from receptor to G protein is more readily achieved for the clonidine/alpha(2)-AR/Goalpha1 complex. Such observations have important implications for receptor theory and drug development.

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.

Recent studies involving a variety of membrane receptors and ion channels indicate that diversity exists among these proteins as evidenced by tissue-specific and developmentally related expression of different isoforms. Alpha 2-Adrenergic receptors, plasma membrane proteins involved in sympathetic neurotransmission, may similarly represent a nonhomogeneous class of binding sites based on the following observations. First, their activation can elicit a wide variety of effector cell responses, which are apparently triggered by at least three different signal transduction mechanisms. Second, alpha 2-adrenergic receptors in various tissues and species exhibit marked differences in their ligand recognition properties. To determine if heterogeneity of the receptor protein itself is involved in generating this diversity, we structurally characterized the alpha 2-adrenergic receptor in two tissues that exhibit the greatest differences in ligand recognition properties, neonatal rat lung and human platelet. We report here that these differences in ligand recognition are maintained after partial receptor purification (50-100-fold) and are associated with distinct differences in the physical and structural properties of the receptor protein. The human platelet and neonatal rat lung receptor differ in the apparent molecular weight of their hormone-binding subunits (human platelet, Mr approximately 64,000 versus neonatal rat lung, Mr approximately 44,000) as well as in the number or type of their associated oligosaccharide moieties. The observed diversity is consistent with expression of isoforms of the alpha 2-adrenergic receptor and suggests the presence of more than one gene encoding similar but distinct receptor proteins.

α2-Adrenergic receptors recognize a number of molecules with diverse chemical structures, including the yohimban diastereoisomers yohimbine and rauwolscine, catecholamines, guanidinium analogs, and imidazolines, such as clonidine. The affinity of the receptor protein for some of these ligands can vary by 10–100-fold among various tissues and species, suggesting a heterogeneous class of binding sites. Certain cellular effects elicited by the compounds possessing an imidazoline or guanidinium moiety may actually be mediated by a membrane receptor distinct from the α2-adrenergic receptor. To determine whether this imidazoline/guanidinium receptive site (IGRS) and the α2-adrenergic receptor represent distinct proteins, we solubilized and partially characterized the two binding sites in rabbit kidney. This tissue expresses both α2-adrenergic receptors and high affinity imidazoline/guanidinium binding sites, the latter which are rauwolscine-insensitive but can be identified with the benzodioxan [3H]idazoxan. The IGRS and α2-adrenergic receptor in rabbit kidney exhibit distinct ligand recognition properties, which are maintained after solubilization and partial purification. In addition, the two receptors can be physically separated by heparin-agarose or lectin affinity chromatography indicating that the two binding sites are distinct entities. [3H]Idazoxan binding is trypsin-sensitive, indicating that the IGRS is a protein rather than a lipid component of the plasma membrane. [3H]Idazoxan binding is not inhibited by endogenous agonists for known neurotransmitter receptors. However, the IGRS does recognize clonidine-displacing substance, a small non-catechol compound isolated from calf brain, suggesting the existence of a previously uncharacterized hormonal/neurotransmitter receptor system.

As an initial approach to define the regulatory elements and transcriptional factors that account for cell-restricted expression of the alpha2c-adrenergic receptor (AR) gene, we isolated and characterized the receptor gene and identified regions of the gene conferring cell-specific expression. A 4300-nucleotide (nt) fragment of the 5'-flanking region of the rat alpha2c-AR gene was isolated from a genomic library. The genomic sequence contained the uninterrupted sequence of the 5'-untranslated region of a previously isolated alpha2c-AR cDNA clone indicating the lack of introns in the 5' gene segment. RNase protection assays and/or RNA blot analysis indicated the expression of alpha2c-AR mRNA in rat brain but not in kidney or liver, which is consistent with the major expression of this gene in neuronal tissue. The 5' gene segment was used to identify sites of transcriptional initiation and promoter activity by RNase protection assays and transient transfection of reporter gene constructs. With the use of RNA probes progressively upstream of the translational start site, RNase protection assays with rat brain total RNA indicated multiple sites of transcriptional initiation within a approximately 70-nt span (-660 to -730 nt 5' to the translational start codon). The zone of transcriptional initiation was part of a larger GC-rich area of the 5' gene segment that is a characteristic of genes initiating transcripts at multiple sites. The promoter activity of this zone of transcriptional initiation and the influence of gene segments 5' to this area were addressed using chloramphenicol acetyl transferase reporter gene constructs. Transient transfection of reporter gene constructs indicated that a 96-nt gene fragment (-699/-603 relative to the translational start codon) was sufficient to direct transcription in the neuroblastoma X glioma hybrid cell line NG108-15, a cell line expressing the endogenous alpha2c-AR. Promoter activity was not observed in constructs lacking the zone of transcriptional initiation. The promoter segment was inactive when introduced into the rat glioma cell line C6B4, the rat submandibular cell line RSMT-A5, and the rat pancreatic beta cell line RIN-5AH, all of which do not express the endogenous alpha2c-AR gene. Upon incubation with nuclear extracts, a 129-nt fragment encompassing the promoter exhibited a gel mobility shift pattern that was specific for cells expressing the receptor protein and involved a nuclear protein that recognized a Sp1 oligonucleotide. These data indicate that a 96-nt gene promoter segment of the alpha2c-AR gene functions in a cell-type-specific manner.

Imidazoline/guanidinium receptive sites (IGRS) belong to a family of membrane proteins that selectively recognize certain pharmacologically active compounds with an imidazoline or a guanidinium moiety. The role of such proteins in the cellular responses elicited by these compounds is unclear, but two members of this protein family are identical to isoforms of monoamine oxidase, an enzyme involved in the metabolism of monamine neurotransmitters. To characterize the structural and ligand recognition properties of the imidazoline binding proteins, we used the photoaffinity adduct [125I]iodoazidophe-noxymethylimidazoline ([125I]AZIPI) to label their ligand binding subunits in selected target tissues (kidney, pancreatic B cells, liver, and salivary gland). Photoaffinity labeling of membrane preparations or subcellular particulate fractions from various rat, rabbit, or hamster tissues indicated two labeled peptides of M(r) approximately 55,000 and approximately 61,000, the relative tissue distribution of which mirrored the expression of the A or B isoforms of monoamine oxidase. The ligand binding subunit of imidazoline binding proteins was identified on two peptides of M(r) approximately 55,000 and approximately 61,000 in rat and rabbit kidney, rat liver, rabbit salivary gland, and the pancreatic B cell line RIN-5AH, whereas only an M(r) approximately 61,000 peptide was observed in rat salivary gland and the hamster pancreatic B cell line HIT-T15. Saturation labeling experiments indicated that [125I]AZIPI exhibited similar affinity (Kd approximately 2-3 nM) for both the M(r) approximately 55,000 and approximately 61,000 peptides. However, competitive inhibition of photolabeling indicated that the two peptides were distinguished by their affinity for the guanidinium guanabenz or their interaction with potassium. Although some types of imidazoline binding sites are located on the enzyme monoamine oxidase, the nonisoform selective enzyme inhibitor pargyline did not alter photoaffinity labeling of either the M(r) approximately 55,000 or approximately 61,000 peptide, indicating that imidazolines/guanidiniums and active site inhibitors of monoamine oxidase interact with different domains on the enzyme. In rat kidney and liver, an additional photolabeled peptide of M(r) approximately 25,000 was observed, and its ligand recognition profile was distinct from the M(r) approximately 55,000 and approximately 61,000 species. In contrast with the mitochondrial location of the larger peptides, subcellular fractionation of liver homogenates indicated that the M(r) approximately 25,000 localized to the plasma membrane.

Imidazoline/guanidinium receptive sites (IGRS) are membrane proteins that exhibit high affinity for various compounds with an imidazoline or guanidinium moiety. The structure of these binding sites and their significance in the broad pharmacological action of such ligands are unclear. To address this issue, we developed selective high affinity compounds that could be radioiodinated and used as molecular probes for structural characterization of these proteins. This report describes the synthesis and characterization of such a molecule, 2-(3-amino-4-[125I]iodophenoxy)methylimidazoline ([125I]AMIPI). [125I]AMIPI is structurally related to cirazoline, an imidazoline exhibiting high affinity for IGRS and the family of related imidazoline binding sites. The phenyl-substituted analogue of cirazoline, 2-(3-aminophenoxy)methylimidazoline, was generated by alkylation of acetamidophenol with 2-chloromethylimidazoline. 2-(3-Aminophenoxy)methylimidazoline exhibited high affinity for IGRS in rabbit kidney membranes, as determined in competition binding studies with [3H]idazoxan (Ki = 12.5 +/- 7.5 nM), and was radioiodinated by chloramine-T oxidation to yield [125I]AMIPI. The binding properties of [125I]AMIPI were determined in membranes prepared from two representative tissues, rabbit kidney cortex and rat liver. Specific binding of [125I]AMIPI was saturable and of high affinity, as determined by Scatchard analysis of saturation binding isotherms (rabbit kidney, Kd = 2.0 +/- 0.9 nM, Bmax = 554 +/- 201 fmol/mg, five experiments; rat liver, Kd = 2.6 +/- 1.3 nM, Bmax = 73 +/- 10 fmol/mg, three experiments).(ABSTRACT TRUNCATED AT 250 WORDS)

The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-4-amino-phenethyl]carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-[N-4-aminophenethyl]carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand [3H]rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC [17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -[125I]iodophenethyl)carboxamide], was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM). In competition studies, alpha-adrenergic antagonists and agonists inhibited the binding of 125I-rau-pAPC with a potency order consistent with an interaction at alpha 2-adrenergic receptors (rauwolscine greater than phentolamine greater than prazosin; clonidine greater than (-)-epinephrine greater than (-)-norepinephrine greater than dopamine greater than (+)-epinephrine). In rat liver and human platelet membranes, high affinity binding of 125I-rau-pAPC was also observed (liver, KD = 1.2 +/- 0.4 nM; platelet, KD = 3.2 +/- 1.5 nM). In addition, the density of alpha 2-adrenergic receptors identified from binding studies with 125I-rau-pAPC in kidney, liver, and platelet membranes was similar to that observed in parallel studies with [3H]rauwolscine. These findings indicate that 125I-rau-pAPC is a high affinity probe that selectively identifies alpha 2-adrenergic binding sites. Availability of this radioligand should facilitate the localization and biochemical characterization of this alpha-adrenergic receptor subtype.

In hepatocytes freshly isolated from adult female rat livers, catecholamine-stimulated glycogenolysis is mediated predominantly by alpha 1-adrenergic receptors, and to only a minimal extent by beta 2 receptors. Primary cell culture of these hepatocytes results in a switch in the adrenergic control of glycogenolysis from an alpha 1 to a predominant beta 2 type of response. To investigate whether this switch is due to an alteration in the plasma membrane receptor composition, we characterized alpha 1 and beta 2-adrenergic receptors in both freshly isolated and cultured hepatocytes, using radioligand-binding techniques. Binding of the selective alpha 1-adrenergic antagonist [3H]prazosin and the beta-adrenergic antagonist [125I]pindolol to intact freshly isolated hepatocytes was of high affinity, saturable, and of appropriate specificity for an alpha 1- and beta 2-adrenergic receptor, respectively. Equilibrium binding studies evaluated by a computer-assisted curve-fitting procedure indicated interaction with a single class of high affinity sites for radiolabeled prazosin (KD = 126 +/- 10 pM; Bmax = 93,000 +/- 5,500 sites/cell) and pindolol (KD = 66 +/- 6 pM; Bmax = 2,000 +/- 700 sites/cell). In intact hepatocytes and in membranes prepared from these hepatocytes, competitive inhibition curves revealed the coexistence of two different sites with high and low affinities for agonists at both alpha 1- and beta 2-adrenergic receptors. When isolated hepatocytes were kept in monolayer cell culture for up to 72 hr, the switch in adrenergic control of glycogenolysis (phosphorylase a activation) from an alpha to a beta pathway was confirmed and was associated with a progressive decrease in the number of alpha 1 receptors and an increase in beta 2-adrenergic receptor density, without marked change in the affinity of agonists or antagonists. To investigate the mechanism(s) of this reciprocal change, a number of perturbations were examined including alterations in the composition of the culture medium and the influence of various hormones and inhibitors of cellular function. De novo protein synthesis is implicated in both receptor alterations as the inhibitors cycloheximide and actinomycin D prevented the increase in beta- and attenuated the decrease in alpha-adrenergic sites. The other perturbations were without effect. Thus, these studies provide evidence for a coupling of the functional alteration in glycogenolysis to changes at the receptor level per se. The mechanism underlying the reciprocal changes in hepatocyte adrenergic receptors during culture remains undefined.(ABSTRACT TRUNCATED AT 400 WORDS)

To define the integration of multiple signals by different types of adenylyl cyclase (AC) within the cell, we altered the population of enzymes expressed in the cell and determined the subsequent processing of stimulatory and inhibitory input. DDT1-MF2 cells expressed AC VI-IX and were stably transfected with AC II, III, or IV. Enzyme expression was confirmed by RNA blot analysis and functional assays. Basal enzyme activity was only increased in AC II transfectants (6-fold). Maximum stimulation of enzyme activity was increased in each of the AC transfectants to varying extents. alpha2A/D-AR activation elicited enzyme type-specific responses. alpha2-AR activation inhibited the effect of isoproterenol in control transfectants, and this action was magnified in AC III transfectants. In AC II and AC IV transfectants, alpha2-AR activation initiated both positive (Gbetagamma) and negative signals (Gialpha) to the Gsalpha-stimulated enzyme, and both types of signals were blocked by cell pretreatment with pertussis toxin. The negative input to AC II from the alpha2-AR was blocked by protein kinase C activation in AC II transfectants, but it was the positive input to AC IV that was compromised by protein kinase C activation. These data indicate that the integration of multiple signals by adenylyl cyclases is a dynamic process depending upon the enzyme type and phosphorylation status.

We report that two subtypes of alpha2-adrenergic receptors (alpha2A/D- and alpha2C-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, alpha2C-AR; coding region plus 1140 nt, alpha2A/D-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein. 90% of the NIH-3T3 alpha2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha2A/D-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha2C-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha2C-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a key mechanism for regulating alpha2C-AR expression and associated signaling events.

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208-Arg387), M3-MR (Gly308-Leu497), or alpha2A/D-adrenergic receptor (AR) (Lys224-Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3 fusion protein, but not to the control GST peptide or i3 peptides derived from the alpha2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified beta-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]beta-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction of in vitro translated [3H]beta-arrestin, [3H]visual arrestin, and [3H]beta-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of beta-arrestin with the M3-MR peptide required both the amino (Gly308-Leu368) and carboxyl portions (Lys425-Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3.

Purified bovine brain G-protein was used in a solution phase assay to identify membrane-associated proteins that influenced the activation of heterotrimeric G-proteins. Detergent-solubilized membrane extracts from the neuroblastoma-glioma cell hybrid NG108-15, but not the parent C6B4 glioma cell line, increased [35S]GTPgammaS binding to purified G-protein by approximately 460%. The G-protein activator was heat-sensitive, and the magnitude of its action was related to the amount of extract protein. The biophysical and biochemical properties of the G-protein activator were determined using DEAE ion exchange chromatography, gel filtration, and a lectin affinity matrix. In the presence of added GDP (1 microM), the enriched G-protein activator increased the initial rate of [35S]GTPgammaS binding to brain G-protein by up to 4-fold. In the absence of added GDP, the G-protein activator elicited an initial burst in [35S]GTPgammaS binding to brain G-protein within the first 30 s, after which the rate of nucleotide binding to G-protein was similar in the absence or presence of the G-protein activator. The stimulation of nucleotide binding to brain G-protein by the activator was also observed after resolution of Galpha from Gbetagamma. The G-protein activator was distinct from other proteins (neuromodulin, tubulin, and beta-amyloid precursor protein) that influence nucleotide binding to G-protein, indicating the existence of a novel signal accelerator.

Monoamine oxidase B (MAO-B) was recently identified as a member of the family of imidazoline binding proteins. To localize the imidazoline binding domain on MAO-B, we labeled the domain with the imidazoline photoaffinity adduct [125I]2-(3-azido-4-iodophenoxy)methylimidazoline in rat and human liver and visualized labeled peptides by autoradiography/sodium dodecyl sulfate-polyacrylamide gel electrophoresis after CNBr cleavage of the labeled protein. Based on species-specific fragmentation patterns and immunoprecipitation of labeled peptides, the imidazoline binding domain was localized to residues K149 to M222 of human MAO-B. The imidazoline binding domain is encompassed within a region that influences substrate processing but is distinct from primary sites of interaction for the enzyme inhibitors pargyline and lazabemide (Ro 19-6327). Radioligand binding assays and photoaffinity labeling also indicated that the various classes of compounds did not cross-compete at the different enzyme domains. Identification of an imidazoline binding domain on MAO-B provides a new opportunity for the potential pharmacological development of imidazoline/guanidinium compounds and also presents additional avenues for structure/function analysis of the monoamine oxidase enzymes.

α2-Adrenergic receptors (α2-AR) are negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to α2-AR agonists, suggesting that the receptor can couple to other signal transduction pathways. One potential pathway may be the stimulation of Na+/H+exchange elicited by α2-AR activation in renal proximal tubule cells, platelets, and the NG-10815 cell line. To determine whether the various receptor-effector coupling mechanisms operate in a tissue-specific manner, we studied the effect of α2-AR activation on basal and stimulated Na+/H+exchange in epithelial cells isolated from human colon (HT-29 adenocarcinoma cells). Na+/H+exchange was measured by quantitation of intracellular hydrogen ion concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and 22Na+uptake. HT-29 cells expressed an amiloride-sensitive Na+/H+exchanger that was activated by reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observed after acid loading (0.57 ± 0.07 pH units/min/104cells) was dependent on external sodium and was blocked by amiloride (Ki∼ 2.1 µM). Although epinephrine and the selective α2-AR agonists Clonidine and UK-14304 inhibited forskolin-activated adenylylcyclase, these compounds did not alter basal Na+/H+exchange. Stimulated Na+/H+exchange was similarly unaffected by epinephrine. In contrast, stimulated Na+/H+exchanger activity was completely inhibited by the selective α2-agonists clonidine, UK-14304, and guanabenz. This inhibitory effect was not blocked by the α2-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchanger molecule itself. Structure/activity studies indicated that the compounds inhibiting exchanger activity possess either an imidazoline or guanidinium moiety. Although these molecules bear structural similarity to amiloride, they did not inhibit the amiloridesensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as “amiloride-like” ligands selective for the Na+/H+exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, α2-adrenergic receptors are not coupled to the Na+/H+exchanger, suggesting that the cell-signaling mechanisms utilized by the α2-AR are tissue specific.

The binding subunit of the alpha 1-adrenergic receptor has been identified as an Mr = 80,000 peptide in several tissues. Adsorption of the alpha 1-adrenergic receptor to a wheat germ agglutinin lectin-agarose resin suggests that the receptor protein is glycosylated. In this study, we investigated the nature of the carbohydrate chains linked to the alpha 1-adrenergic receptor peptide. The alpha 1-adrenergic receptor from DDT2 MF-2 smooth muscle cell and rat brain membranes was photolabeled with 125I-azido-prazosin [( 125I]CP65,526) and then treated with exoglycohydrolases prior to SDS-PAGE and autoradiography. Removal of terminal sialic acid residues by neuraminidase decreased the receptor Mr by 6,000; however, alpha-mannosidase was without effect, indicating complex type glycosylation of the receptor-protein. Similar results were observed for the rat hepatic membrane alpha 1-adrenergic receptor. Removal of N-linked carbohydrates at asparagine residues by peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase (from Flavobacterium meningosepticum) resulted in a specifically labeled peptide at Mr = 50,000-55,000 in DDT1 MF-2 membrane and solubilized receptor preparations. Treatment of DDT1 MF-2 cells with swainsonine or (+)-1-deoxymannojirimycin, inhibitors of complex type carbohydrate chain biosynthesis, caused a reduction in the apparent molecular weight of the receptor (Mr = 60,000) but did not alter the number of alpha 1-adrenergic receptors per cell or their affinity for the radioligand [3H]prazosin. These findings indicate that the alpha 1-adrenergic receptor is heavily glycosylated, the major oligosaccharide moiety being of the complex type, N-linked to asparagine residues. The peptide backbone of the receptor has an Mr less than or equal to 55,000, consistent with the predicted molecular mass of other membrane neurotransmitter receptors based on sequence analysis of isolated cDNA clones.