Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Coupling of the α1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site

At rat hepatic membrane α₁-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in α₁-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca²⁺-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (−pi), or in −pi membranes treated with Ca²⁺ (−pi, +Ca²⁺). In these various membrane preparations, different M_r forms of the receptor are also identified by photoaffinity labelling with [¹²⁵I]CP65 526, an aryl azide analog of the α₁-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant M_r = 80 000 subunit is identified in +pi membranes, in −pi membranes a proteolytic M_r = 59 000 fragment is also observed. In −pi, +Ca²⁺ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [¹²⁵I]CP65 526 labelling was determined by laser densitometry scanning and computer analysis. At the M_r = 80 000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in −pi membranes. By contrast, agonist-binding at the M_r = 59 000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved M_r = 59 000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the α₁-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

ADP-ribosylation of brain synaptosomal proteins correlates with adenylate cyclase activation

ADP-ribosylation of the adenylate cyclase $\text{G}\text{F}$ regulatory subunit by cholera toxin is a major tool for the study of this enzyme. Investigation of the brain enzyme has been hampered up to now by the failure to demonstrate cholera toxin-dependent ADP-ribosylation of membrane-bound proteins. Synaptosomes prepared by flotation from fresh brains homogenized in the presence of protease inhibitors yielded membranes of which several proteins could be ADP-ribosylated by the toxin. The same membranes subjected to mild proteolysis could not be ADP-ribosylated. Adenylate cyclase activation and ADP-ribosylation were simultaneous processes. The major labeled species was of 47,000 M_r. It was solubilized by Lubrol-PX, together with other labeled polypeptides. As analyzed on sucrose gradients, the 47,000 M_r protein was found both in the 3S region, and in the adenylate cyclase containing fraction (9.1S).     

The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

β-adrenegic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0–8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the β-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na⁺K⁺(Mg²⁺)-ATPase activity and a 6–8-fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2–3-fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological β₂ type response with isoproterenol (1.8 · 10^?8 M) > epinephrine (1.1 · 10^?7 M) > norinephrine (3.2 · 10^?6 M). In contrast, binding studies employing (?)-[³H] dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (?)-[³H]dihydroalprenolol by catecholamine agonists.Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of M_r 80 000 and 86 000, whereas in the adult only a single M_r 133 000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.     

Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.     

Pancreatic B-cells secrete a range of novel peptides besides insulin

Insulin secretion by a transplantable rat islet B-cell tumour is accompanied by the release of two putative proinsulin cleavage intermediates, four peptides of M_r 9000–12 000 (excluding proinsulin) and peptides of M_r 21 000, 34 000 and 60 000. Granule-enriched subcellular preparations contain major peptides of identical M_r values. Of these peptides seven at least coincide in molecular weight with peptides secreted by isolated rat islets and thus may be constituents of the normal insulin secretory granule.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

Evidence for two types of β-adrenergic-sensitive adenylate cyclase activities in bovine cerebellum

A latent, as well as an expressed form of adenylate cyclase coupled to β-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [³H]adenine labeling and was found to be coupled to a β₁-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same with [³H]ATP was stimulated via β₂-adrenergic receptors.     

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

II. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung

β-Adrenergic receptors were identified in membrane fractions of fetal and postnatal rat lung with the β-adrenergic antagonist (?)?[³H] dihydroalprenolol, (?)?[³H] DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to day 28 of postnatal life, 46±7 to 491±69 femtomole·mg^?1 protein. Neither the K_D, approximately 0.8nM for [³H]DHA, nor the β-adrenergic subtype changed with age. Classical agonists competed for the β-receptor with properties characteristic of β₂-adrenergic binding. Analysis of the inhibition of receptor binding by selective β-adrenergic agents demonstrated approximately 75% β₂ and 25% β₁ β-adrenergic subtypes in fetal rat lung membranes. The increase in β-adrenergic receptor during development was associated with adenylate cyclase activity which was sensitive to catecholamines at all ages studied, supporting the possible role of the β-adrenergic receptor system in the postnatal regulation of pulmonary function.     

Fractionation by velocity sedimentation of Torpedo nicotinic post-synaptic membranes

Velocity sedimentation on sucrose gradients containing Torpedo physiological saline has been utilized to fractionate Torpedo (Torpedo californica and T. nobiliana) post-synaptic membranes isolated initially on the basis of their density by equilibrium centrifugation. Membranes are separated into two populations: (1) those retained within the gradient (referred to as gradient pool); and (2) membranes sedimenting rapidly through the gradient (referred to as f 22, fraction 22 of the gradient). Comparison of their polypeptide compositions by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicates that the gradient pool consists of highly purified nicotinic post-synaptic membranes containing the peptides of the acetylcholine receptor and a peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000, while f 22 contains the contaminating membranes present in the initial suspension as well as a small fraction of the nicotinic post-synaptic membranes. On the basis of the kinetics of efflux of ²²Na⁺ from the membrane fractions, it is concluded that the gradient pool contains most of the sealed vesicles with functional nicotinic receptors. The internal volume (μl/mg protein) of those membranes exceeds that of f 22 by a factor of 4, and greater than 85% of that internal volume is equilibrated by the nicotinic agonist carbamylcholine, while for f 22 only 40% is equilibrated. Thin-section electron microscopy has been used to estimate the distribution of vesicle sizes. The observed distribution for the gradient pool indicates that these vesicles are a size homogeneous population of diameter 0.3 μm, while f 22 contains a number of smaller and larger vesicles. Torpedo post-synaptic membranes have been treated with alkali to remove the non-receptor peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000. After alkaline extraction, velocity sedimentation permits the isolation of a population of size-homogeneous and well-sealed vesicles containing only the peptides of the nicotinic receptor. It is concluded that upon homogenization, the innervated surface of the Torpedo electroplax tends to form vesicles of uniform size (0.3 μm) which can be readily isolated by velocity sedimentation and that the peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000 is not required for the maintenance of bilayer structure.     

Differential control of β-endorphin/β-lipotropin secretion from anterior and intermediate lobes of the rat pituitary gland in vitro

The secretion of immunoreactive β-endorphin (β-END_i) and β-lipotropin (β-LPH_i) by neurointermediate lobes (NIL) and anterior lobe (AL) cells of the rat pituitary gland was studied in an $\text{in vitro}$ superfusion system. Peptides were characterized by gel chromatography on Sephadex G-50 and by two radioimmunoassays: a β-LPH assay in which β-END did not crossreact (β-LPH_i) and a β-END/β-LPH assay in which β-END and β-LPH showed full crossreactivity (β-END_i/β-LPH_i).$\text{Intermediate lobe}$. The spontaneous secretion of β-END_i/β-LPH_i by the NIL was 1–2 ng/min/lobe. Chromatography showed that 97% of this β-END_i/β-LPH_i eluted at the position of β-END. Dopamine inhibited the spontaneous secretion of β-END and the dopamine-receptor blocker domperidone prevented this inhibition. Isoprenaline caused a 3–4 fold stimulation of the secretion of β-END. The β-adrenergic receptor blocker propranolol abolished this stimulation. Hypothalamic extract, lys-vasopressin, 5-hydroxytryptamine and histamine were ineffective in changing the spontaneous secretion of β-END_i/β-LPH_i.$\text{Anterior lobe}$. The spontaneous secretion of β-END_i/β-LPH_i by AL cells was 0.15–0.20 ng/min/10⁵ cells. Chromatography revealed that about 70% of this material behaved like β-LPH, 30% behaved like β-END. Hypothalamic extract and lys-vasopressin induced a 3–5 fold increase in the secretion of both β-END and β-LPH. Catecholamine, 5-hydroxytryptamine and histamine were ineffective in changing the spontaneous secretion of β-END_i/β-LPH_i.These results indicate that β-END is the predominant β-LPH-related peptide secreted by the intermediate lobe and that its secretion is inhibited via a dopaminergic receptor mechanism and stimulated via a β-adrenergic receptor mechanism. The secretion of β-END and β-LPH by the anterior lobe is not affected by catecholamines but is stimulated by CRF and vasopressin.     

Peptide mapping of fat cell membrane substrates for cholera toxin-catalyzed ADP-ribosylation

Incubating rat fat cell membranes with [³²P]NAD⁺ and cholera toxin results in ADP-ribosylation of three distinct components with approximate molecular weights of 42 000, 46 000 and 48 000. Partial proteolytic peptide maps of the M_r = 46 000 and 48 0000 toxin-specific substrates generated by elastase, α-chymotypsin, or Staphylococcus aureus V-8 protease were nearly identical, while those of the M_r = 42 000 target lacked several peptides common to both of the larger molecular weight targets. In addition, peptide maps generated from the M_r = 42 000 target displayed a number of peptides which were absent from the maps generated from either the M_r = 46 000 or 48 000 targets. These data suggest that the M_r = 46 000 and 48 000 substrates are closely related proteins, however the relationship between the M_r = 42 000 toxin-specific substrate and the larger peptides remains to be established. The relative patterns of fat cell membrane labelling by cholera toxin in the presence of [³²P]NAD⁺     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.