A431 cells incubated with tunicamycin (0.15 micrograms/ml) for 40 h under conditions where incorporation of [3H] leucine into protein was inhibited less than 10% expressed mainly a beta-receptor species of about Mr 40,000 which was ascribed to the nonglycosylated form of the beta-receptor of about Mr 75,000 found in normal A431 cells by photoaffinity labeling. However, the tunicamycin-treated cells expressed the same number of specific beta 2-receptor-binding sites as untreated cells. Moreover, the aglycoreceptors had the same ligand binding properties as beta-adrenoceptors from control cells; but, functional tests of the receptor from tunicamycin-treated cells in reconstituted lipid vesicles showed that receptors from tunicamycin-treated cells had lost coupling efficiency. The coupling defect was at the receptor level since control experiments indicated that the other components of the signal transmission chain from beta-adrenoceptor to adenylate cyclase, the stimulatory regulatory GTP-binding protein of adenylate cyclase and adenylate cyclase, were fully functional. Homologous desensitization in tunicamycin-treated cells was characterized by export from the cell surface and sequestration of about the same number of beta-adrenoceptors as in normal desensitized cells but without further reduction of hormonally stimulated adenylate cyclase below the low level already attained in nondesensitized tunicamycin-treated cells. This was explained by assuming that the receptors removed in the course of homologous desensitization from the surface of tunicamycin-treated cells were already nonfunctional. Thus, beta-adrenergic desensitization in tunicamycin-treated cells is characterized by the functional disengagement of receptor removal and loss of adenylate cyclase activity.

We have previously described a specific protease in turkey erythrocytes that converts the larger 50-kDa (P50) form of the beta 1-adrenoceptor to a smaller 40-kDa (P40) form [Jürss, R., Hekman, M., & Helmreich, E. J. M. (1985) Biochemistry 24, 3349-3354]. Further functional and structural characterization studies of the two forms are reported here. When purified P50 and P40 receptors were compared with respect to their relative capabilities to couple in lipid vesicles with pure stimulatory G-proteins (Gs-proteins) prepared from turkey erythrocytes or rabbit liver, a faster and larger activation of Gs-proteins was observed in response to l-isoproterenol and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with P40 than with P50 receptor. The kon values for P40 were 0.47 min-1 in the case of liver Gs and 0.22 min-1 in the case of erythrocyte Gs, whereas the corresponding values for P50 were 0.34 min-1 and 0.12 min-1, respectively. The binding properties of P50 and P40 forms of the receptor were not different, and desensitization of turkey erythrocytes on exposure to l-isoproterenol did not activate the protease. We furthermore ascertained that only the larger form with a molecular mass of 50 kDa carries the N-linked carbohydrates, which are removed on proteolytic conversion to the 40-kDa form and have either a triantennary or a tetraantennary nonfucosylated complex-type structure containing terminal sialyl residues.

Mammalian β2-adrenergic receptors (R) have been shown to be structurally heterogenous with respect to glycosylation (Stiles et al. J. biol. Chem.259, 8655 (1984)). They are also heterogenous with respect to functional coupling to Ns. The ternary H.R.Ns complex can be frozen in the presence of the alkylating reagent N-ethylmaleimide. In hamster lung membranes 45% of the receptors are agonist/N-ethylmaleimide sensitive (i.e. coupling-prone receptors). β-Receptors in both native and isoproterenol/N-ethylmaleimide pretreated membrane preparations are retained by affinity chromatography on concanavalin A and wheat germ agglutinin and are equally sensitive to neuraminidase treatment. This is exhibited by the increase in mobility of the 125I-iodocyanopindolol-azide photoaffinity labeled receptor peptide in SDS-polyacrylamide gel electrophoresis. These observations suggest that there is no link between the structural and functional heterogeneity of the receptors. Moreover, both partial (using neuraminidase) and near total (using endoglycosidase F) deglycoslation of membrane-bound receptors does not affect the H.R.-Ns coupling capacity as compared to native receptors.

A protease that can be inhibited by glutathione, dithiothreitol, and o-phenanthroline but not by ethylenediaminetetraacetic acid converts the 50-kilodalton beta-adrenergic receptor in turkey erythrocyte membranes to a 40-kDa polypeptide which retains the specific ligand binding site. This conversion is attenuated in intact erythrocytes. The large 50-kDa peptide contains N-linked, complex carbohydrates and is retained on wheat germ agglutinin-Sepharose. The 40-kDa product of proteolysis does not bind to the wheat germ agglutinin and can thus be separated from the 50-kDa polypeptide by lectin chromatography. However, the large difference in molecular weights of the two receptor peptides cannot be accounted for solely by the different extent of glycosylation.

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N-7-(4-nitrobenzo-2-oxa-1,3-diazolyl)-omega-amino-n-alkanoic acid beta (N,N,N-trialkylammonium) ethylesters (NBD-n-acylcholines) and report here on their physiological and biochemical properties. All NBD-n-acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length. The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n = 4 and n = 3. The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 10(8) M-1 S-1 and do not vary significantly with the chain length of the NBD-n-acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length. The quenching of fluorescence of NBD-n-acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the omega-amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein. With their advantageous fluorescence properties, their simple pharmacology, and their clear structure-function relationships, these compounds are useful tools for the study of cholinergic mechanisms.

We have studied the interaction of N-7-( 4-nitrobenzo-2-oxa-1,3-diazole)-o-aminoalkanoic acid b-(N-trimethylammonium) ethyl esters (NBD-n-acylcholines) with high and low molecular weight acetylcholine esterase from Electrophorus electricus. 1) NBD-n-acylcholines competitively inhibit enzymatic hydrolysis of acetylthiocholine (Ki of the order of 5 x lo-' M) but are hydrolyzed by the esterase several ' The abbreviation used is: NBD, N-7-(4-nitrobenzo-2-oxa-1,3-dia-' R. Riichel, D. Watters, and A. Maelicke, unpublished data.

We have studied the properties of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-omega-aminohexanoic acid beta-(N-trimethylammonium)ethyl ester, a fluorescent analog of acetylcholine at the cellular level by using pharmacological and electrophysiological techniques and at the molecular level by measuring the kinetics of interaction with solubilized acetylcholine receptor and with acetylcholine esterase (EC 3.1.1.7). The fluorescent drug is a powerful agonist of acetylcholine at the neuromuscular junction and also strongly desensitizes muscle fibers. Interaction with acetylcholine receptor is accompanied by large changes in the drug's fluorescence. From the kinetics of interaction studied by means of a stopped-flow fluorimeter with laser light source, we obtained a second-order forward rate constant in excess of 1 X 10(8) M-1 sec-1 and an initial dissociation rate constant (k1) of 0.5 sec-1 for receptor from Electrophorous electricus. Interaction of this analog with acetylcholine esterase from E. electricus is accompanied by a transient decrease in fluorescence followed by an increase leading to a stable plateau value at a level near the original one. The initial decrease in fluorescence followed second-order kinetics with k2 of the order of 10(9) M-1 sec-1. The slower consecutive reaction which could be blocked by phosphorylation of the esteratic site, was of first order with k1 = 0.05 sec-1.