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The human cannabinoid receptor associated with the CNS (CB1) binds Δ9‐tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein‐coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys192 of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild‐type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP‐55,940 and the aminoalkylindole WIN 55,212‐2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP‐55,940, whereas WIN 55,212‐2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand‐induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212‐2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP‐55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212‐2 binding, the data suggest a role for Lys192 in WIN 55,212‐2‐induced receptor activation.

The human cannabinoid receptor CB1 functionally couples primarily to Gi-, but also to Gs-mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl-terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist-independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs- and not a Gi-mediated pathway. Treatment with the CB1-specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP-55,940 to the L341A/A342L receptor was not markedly different from that for the wild-type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

The question of the mode of action of the E. coli supernatant factors T" 2 (Tu and T.)3 and GI 2, 4 has remained open until recently. It is now clear that the T factor, together with GTP, participates in the attachment of aminoacyltRNA's to ribosomes.5'-After the binding reaction, peptide bond formation ensues if the peptidyl site carries peptidyl-tRNA,8 or an aminoacyl-tRNA with a free6' 9 or a blocked6' 7 a-amino group on the amino acid. Polymerization ceases at this stage and continues only upon addition of G factor. It has been proposed by Nishizuka and Lipmann'0 that GTP and the G factor' are involved in messenger RNA movement and, simultaneously, in translocation"2 of the newly synthesized peptidyl-tRNA from the aminoacyl to the peptidyl site. The completion of this process opens the aminoacyl site for entry of the next aminoacyl-tRNA. This possibility has been examined by various laboratories. Using a mammalian system, Skogerson and Moldave8 have shown that aminoacyl transferase II (analogous to G factor in mammalian systems) and GTP lead to the transposition of peptidyl-tRNA from the aminoacyl to the peptidyl site. The work of Erbe and Leder7 indicates that whereas only T and GTP are required to form F-Met-Phe-tRNA with AUG (U6) as template, the translation of the third triplet codon depends on G.In this paper we elaborate further on the T-dependent binding of aminoacyltRNA to ribosomes and show (in agreement with Skogerson and Moldave8) that if GTP is replaced by GMP-PCP, the aminoacyl-tRNA is bound, but subsequent peptide bond formation is prevented with prebound N-acetylPhe-tRNA. The aminoacyl-tRNA bound in this way does not interfere with the puromycin release of the prebound N-acetylPhe.We also describe experiments which concern the possible role of the G factor in polymerization. By stepwise addition of aminoacyl-tRNA's to ribosomes, we have synthesized N-acetyltriPhe-tRNA, and demonstrate that after one peptide has been formed, an incubation period with G factor and GTP is required before the peptide chain can be lengthened by another amino acid. A similar approach to this problem was undertaken by Schweet and his collaboratorsl3' 14 in a reticulocyte cell-free system.Methods.-Preparation of ribosomes and supernatant factors: E. coli B cells were harvested in mid-log phase2 and were used to prepare ribosomes as reported previously.6 The supernatant factors T (T. and T.) and G were isolated from Ps. fluorescens, and the T fraction was separated into a T. peak, a T.T. peak, and a peak enriched in T, by chromatography on a DEAE-cellulose column as described by Lucas-Lenard and Lipmann."5 Acetylation of phenylalanyl-tRNA: C"4-Phe-tRNA was acetylated as described byHaenni and Chapeville.-6 Binding of N-acetyl-C'4-Phe-tRNA and of HS-Phe-tRNA to ribosomes; analysis of the products: The reaction mixture (incubation I) contained, in a total volume of 1 ml: 50Amoles of Tris-HCl, pH 7.4, 10 Mmoles of magnesium acetate, 160 ,Amoles of NH4Cl, 10 Mmoles of DTT, 40 /Ag of poly U, and approximately 9.6 A260 units ...

Two fractions which complement the ribosomes for amino acid polymerization were obtained by DEAE-Sephadex chromatography of E. coli extracts.'-3 Using a stepwise procedure,' the fraction eluting first was heat-stable; the more retarded fraction, which coincided with a ribosome-dependent GTPase,2 3 was heat-labile. However, when a linear salt gradient was employed for elution,3 it was the earlier fraction that turned out to be unstable and the later stable.In an attempt to solve the paradox of this switch in stability, the polymerizing factors were isolated from Pseudomonas fluorescens. A different microorganism was chosen with the hope that new facets of the problem of protein synthesis might be exposed, and that some possible advantages in the stability or ease of isolation of the factors might be observed. Using Ps. fluorescens and E. coli factors interchangeably, it was found that the T fraction3 could be split into a stable component, T8, and an unstable component, Tu.4 Tu is very heat-labile and, depending on the elution procedure, can appear associated with the earlier or the later DEAE-Sephadex eluates.Materials and Methods.-Pseudomonas fluorescens, NCIB 8248, was grown on a synthetic medium as described by Wade and Robinson.6 The cells were harvested in log phase, washed twice with 0.01 M Tris-HCl buffer, pH 7.4, and 0.01 M MgCl2, and stored at -15°as a paste. E. coli B was grown as described.6 E. coli soluble RNA (General Biochemicals, Chagrin Falls, Ohio) was charged with radioactive phenylalanine according to Conway,7 using 0.6 JAmole of C'4-L-phenylalanine, specific activity 351 gc/,gmole. The specific activity of the charged sRNA was 0.46 ,gmole C'4-Lphenylalanine per ,ug sRNA.Preparation of ribosomes: In most of the experiments, E. coli ribosomes were employed because they were more stable to extensive salt washes than those from Ps. fluorescens; otherwise, the ribosomes were completely interchangeable.Ribosomes from E. coli B were isolated by a modification of the method of Nishizuka and Lipmann.3 After sedimentation at 105,000 X g, they were washed in 0.01 M Tris-HCl buffer, pH 7.4, 0.5 M NH4Cl, and 1 X 10-4 M MgCl2. In the subsequent washes, the magnesium coneentration was gradually increased to 1 X 10-3 M in the second, to 0.5 X 10-2 M in the third, and to 1 X 10-2 M in the fourth wash. In the final two washes, the magnesium concentration was maintained but the 0.5 M NH4Cl was omitted. Ribosomes thus isolated were suspended in the buffer mixture of the last wash to give 100 mg/ml. One mg dry weight of the ribosomes is assumed to be equivalent to 14.4 OD units at 260 m1A. This preparation is stable for at least 9 months at -15°; it is free of T. and Tu, but retains a small amount of G (5-8 jujAmoles of p32. phosphate were liberated from GTP32 per 100 gg of ribosomes per 10 min at 300, assayed as described3).For the assay of T. activity, Ps. fluorescens ribosomes, sedimented through 10% sucrose, have proved very useful. They retain an excess of G (1.5 msmoles of P32-phosphate were liberated from G...