©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activating and Inactivating Mutations in N- and C-terminal i3 Loop Junctions of Muscarinic Acetylcholine Hm1 Receptors (*)

(Received for publication, June 14, 1994; and in revised form, December 19, 1994)

Petra Högger Melinda S. Shockley Jelveh Lameh Wolfgang Sadée (§)

From the Departments of Pharmacy and Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The N- and C-terminal junctions of the third intracellular loop (i3) of G protein-coupled receptors play a role in the coupling process. We had previously constructed two triple point alanine mutants of the i3 junction of the muscarinic Hm1 receptor, W209A/I211A/Y212A and E360A/K362A/T366A, which are defective in mediating carbachol stimulation of phosphatidylinositol (PI) turnover (Moro, O., Lameh, J., Högger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276). Each of the corresponding six single point mutations were constructed to determine residues crucial to receptor coupling. Mutants W209A and T366A were similar to or only slightly less effective than wild type Hm1 in stimulating PI turnover. In the N-terminal junction, I211A and Y212A were defective in coupling, and I211A was even more defective than the corresponding triple mutant. Therefore, the triple mutation compensated at least partially for the effect of these two single point mutations. In the C-terminal i3 loop junction, mutant K362A was again more strongly defective than the corresponding triple mutant. In contrast, mutation E360A was found to be activating, leading to elevated PI turnover in the absence of agonist and sensitization toward carbachol activation. Activating mutations in the C-terminal i3 loop junction have been reported previously for the adrenergic receptors, but E360A represents the first muscarinic receptor with substantial basal activity. The effects of the single point mutations observed in this study were not readily predictable from similar mutations from closely related G protein-coupled receptors despite sequence conservation in the i3 loop junctions. Our results caution against defining precise coupling domains in these regions by mutagenesis results.


INTRODUCTION

G protein-coupled receptors (GPCRs) (^1)are organized into three extracellular and three intracellular loops and seven putative transmembrane domains (TMDs). The intracellular receptor domains involved in G protein recognition and activation have been studied extensively. G protein coupling is thought to be mediated by an interaction of several domains, including the second and third intracellular loop (i2/i3 loop)(1, 2, 3) . We recently proposed a highly conserved consensus sequence in the i2 loop, DRYXXVXXPL, as a major coupling domain(4, 5) . However, the i1 loop and the C-terminal tail have also been shown to be involved in receptor coupling(4, 5, 6, 7) .

Mutational analysis, use of synthetic peptides, and construction of chimeric receptors suggest that the N- and C-terminal junctions, proximal to TMD 5 and 6, of the i3 loop play a significant role in G protein recognition and/or activation(1, 8, 9, 10, 11, 12, 13) . In contrast, most of the large middle portion of the i3 loop can be deleted without affecting coupling in case of the human muscarinic receptor subtypes 1, 2, and 3 (Hm1, Hm2, and Hm3)(14, 15) .

The i3 loop is a region of high sequence diversity, and little or no sequence identity of the junctions exists among different GPCR families (16) . However, the i3 loop junctions are conserved among closely related receptors. Thus, a general consensus sequence required for successful G protein coupling has yet to be identified. However, single amino acids have been shown to play important roles in G protein activation in the i3 loop of several GPCRs(11, 12, 17) .

Because of the lack of any obvious consensus sequence for receptor coupling, the structural features of the i3 loop regions affecting coupling were proposed as coupling determinants(10, 11) . Within the i3 junctions, predicted to be alpha-helical, the correct positioning of charged amino acids was thought to be important(10) . However, others found that lipophilic rather than charged amino acids in the C-terminal i3 loop junction determine the ability of the receptor to couple (18) and that amphipathic alpha-helical structural elements are not necessarily required for G protein interaction(19) . Lechleitner et al.(20) were able to switch G protein coupling specificity of m2 and m3 by exchanging a short segment of the i3 loop junction. However, this same segment could be deleted from the Hm1 receptor without affecting coupling(21) . These results raise the question whether the i3 loop junctions directly activate the G protein or whether they serve as hinge regions that control the i3 loop orientation and thereby G protein receptor interactions, or both. However, mutations change both possible contact sites and secondary structures, making it difficult to discern these two possibilities.

Experiments with synthetic peptides derived from receptor regions confirmed the importance of the N- and C-terminal i3 loop junctions for G protein coupling(1, 3, 13, 22, 23) . Peptides derived from these regions either inhibited or stimulated receptor/G protein coupling. However, these results do not prove the location of direct coupling domains, as the peptides may also affect receptor conformation or induce G protein activation nonspecifically (e.g. by polycations or multibasic peptides), which may not represent the physiological activation mechanism.

Several mutations of the C-terminal junction of the i3 loop in adrenergic receptors were found to activate the receptor con-stitutively(24, 25, 26, 27) . These results are consistent with the hypothesis that the receptor is normally in a constrained conformation, which is released to yield active receptors by a conformational change in the i3 loop junctions.

In order to study further the role of the i3 loop in coupling, we prepared several mutants of the N- and C-terminal i3 junctions of Hm1. Previous studies (21) revealed little effect of mutating several residues and regions suspected to play a role in coupling, including residues E214A/E216A/E221A in the N-terminal and K359A/K361A in the C-terminal junction. These results indicate that the proposed G protein activating consensus sequence BBXB or BBXXB (B stands for a basic, X for a non-basic residue)(28, 29) in the N-terminal junction is not applicable in the case of Hm1. We further constructed two triple mutants, W209A/I211A/Y212A (N-terminal junction) and E360A/K362A/T366A (C-terminal junction), which were defective in coupling to PI turnover(4) . If individual amino acids in the junctions directly interact with the G protein we should expect additive effects of the single mutants. However, if the junctions serve as hinge regions, single point mutations may have entirely different effects from the triple mutations, because of compensatory or unpredictable conformational changes. Indeed, two single point mutants were more strongly defective than the triple mutants, and one mutant, E360A, resulted in constitutive activation, suggesting that mutation-induced changes in the i3 loop conformation account at least in part for these results.


EXPERIMENTAL PROCEDURES

Materials

[^3H]NMS (specific activity 85 Ci/mmol) and myo-[^3H]inositol (specific activity 17.7 Ci/mmol) were obtained from Amersham Corp. All other reagents were of analytical grade.

Construction of Vectors Expressing Hm1 Point Mutants

The construction of Hm1 in vector pSG5 was described previously(15) , with EcoRI and BamHI restriction sites at the 5` and 3` ends, respectively. The point mutations were introduced using the ``unique site elimination'' method (Transformer(TM) site-directed mutagenesis kit, Clontech)(30) . All mutations were confirmed by sequencing of the mutated region before further use.

Transfection of Human Embryonic Kidney Cells (U293)

The cells were transfected using calcium phosphate precipitation as described previously(15, 31) . Transient expression yields ranged from 396 ± 149 to 2281 ± 481 fmol/mg protein for wild type Hm1 and for the mutants (Table 1). Cells expressing less than 250 fmol/mg protein were not studied further, because lower yields resulted in decreased maximal PI turnover for the wild type receptor(4) . A marked dependence of coupling efficiency on receptor yield above 250 fmol/mg protein was only observed for mutant Y212A, and dose-response curves were constructed at low and high expression levels of Y212A.



PI Turnover

PI turnover was measured after labeling the cells with 0.2 µMmyo-[^3H]inositol for 24 h(14, 15, 31) . For the assay of inositol monophosphate, which accounts for most of the [^3H]IP activities in the presence of 1 mM LiCl, six-well cell culture dishes (well diameter of 3.5 cm) were used. Results were expressed as percentage of total intracellular ^3H activity, and percentage values were compared between carbachol-treated and untreated cells. Concentrations from 0.1 or 0.3 µM to 10 mM were used for dose-response curves.

To determine the effect of atropine on basal coupling activity of Hm1 WT and E360A, the assay was modified. The standard PI turnover assay involved a 30-min preincubation period with LiCl, followed by an additional 30 min with Li and the test agent. To prevent [^3H]IP accumulation by basal receptor activity during the preincubation, LiCl and atropine were added together, and the incubation was carried out for only 30 min. Under these conditions, atropine was capable of reversing elevated basal [^3H]IP release by E360A close to control levels in nontransfected cells.

Receptor Binding

The assay was performed as described previously(14, 31) . The transfected cells were replated onto 12-well cell culture dishes and allowed to attach overnight. Cells were incubated in phosphate-buffered saline containing 1.5-2 nM [^3H]NMS at 12 °C for 90 min. At the end of incubation, cells were harvested in reaction buffer using a Pasteur pipette, filtered on glass-fiber filters (SS32, Schleicher & Schüll) and rapidly rinsed three times with ice-cold phosphate-buffered saline. Displacement binding curves with carbachol (0.001-100 mM) were performed at a [^3H]NMS concentration of 0.2 nM. The resultant data were fitted using the logistic function B = B(max) - B(min) times L/(IC + L) + NSB, where B is the tracer bound (dpm), L the carbachol concentration, and NSB is the nonspecific binding. The curves were fitted with the MINIM 1.8a program (R.O. Purves, Department of Pharmacology, School of Medicine, University of Otago, Dunedin, New Zealand).


RESULTS AND DISCUSSION

The N- and C-terminal junctions of the i3 loop are nearly identical between m1 and m3 (Fig. 1), suggesting that these domains serve similar functions in both receptors. Deletion of N-terminal amino acids close to TMD 5 caused complete uncoupling of the m3 receptor expressed in Xenopus oocytes(9) , suggesting that the N-junction plays a role in coupling. However, mutation of Glu-214 and Glu-216 had only minor effects on coupling to PI turnover by Hm1(21) . In contrast, the triple point mutant W209A/I211A/Y212A was strongly defective(4) . Therefore, the ability of the corresponding single point mutants to stimulate PI turnover was determined in this study (Table 1). These single point mutants of the i3 junctions vary in their ability to couple to PI turnover. Whereas W209A stimulated PI turnover slightly less than wild type, I211A is strongly and Y212A partially defective. While all three mutants stimulate PI hydrolysis significantly less than the wild type, the coupling defects are not due to reduced agonist affinity, since the IC values for carbachol binding are similar to that of the wild type ( Table 2and Fig. 2). The carbachol (1 mM) induced stimulation of PI hydrolysis by mutant Y212A varied from 20 to 70% of the wild type Hm1 in different experiments, and the efficacy of Y212A paralleled its level of expression. Therefore, carbachol dose-response curves were established for Y212A at a relatively low level (467 fmol/mg protein) and a high level (3470 fmol/mg protein) of expression. The resultant curves gave E(max) values of 17% and 70%, respectively, relative to the WT (Fig. 3A). Furthermore, the EC values were shifted from 7.4 ± 0.1 mM carbachol for WT Hm1 to 48 ± 9 mM for Y212A (high expression curve; the low expression curve gave a similar EC for Y212A, but with a large confidence interval). We previously reported an EC value for Hm1 WT of 8.2 ± 4.0 mM, which is consistent with the value reported here(21) . These results show that the coupling deficiency of Y212A is not profound and can in part be overcome by high expression levels.


Figure 1: N- and C-terminal i3 loop junctions of Hm1 and Hm3. Mutated amino acids of the Hm1 are boxed, amino acids marked with a star were mutated in a previous study(19) . Alignment with Hm3 i3 loop sequence demonstrates that both genes share a high degree of identity. The numbering refers to the Hm1 sequence.






Figure 2: Carbachol displacement curves. Displacement binding curves with carbachol (0.001-100 mM, n = 4 for each concentration, n = 6 for E360A) were performed at a [^3H]NMS concentration of 0.2 nM. Data were normalized for control samples without carbachol. The graph is the average of two to three independent experiments.




Figure 3: Dose-response curves for PI stimulation by wild type Hm1 and mutants Y212A (A) and E360A (B) and non-transfected (naive) cells. Concentrations from 0.1 µM to 10 mM were used for dose-response curves (n = 3-6). The dose-response curve for the wild type Hm1 is similar to that published previously(4, 15, 21) (expression yield 1242 fmol/mg protein). For the Y212A dose-response curve (panelA), the expression of the mutant was 467 and 3480 fmol/mg protein for the low expression and high expression curves, respectively. The EC for the high expression curve was 48 ± 9 µM. Expression yields were 873 and 760 fmol/mg protein for E360A, in the two different experiments averaged in panelB. The carbachol EC values were 7.4 ± 1.0 µM for Hm1 wild type and 1.17 ± 0.89 µM for E360A (mean ± S.D. of three independent experiments).



The I211A mutant was more defective in coupling to PI turnover (range 12-20% relative to wild type) than the triple mutant W209A/I211A/Y212A (p leq 0.01), even though the level of expression was similar (Table 1). Therefore, the defects observed with the single point mutations were not additive in the triple mutant. This result supports the hypothesis that the mutations affect the secondary structure of the i3 loop junction. Consequently, the introduction of multiple mutations could have partially compensated for any structural disturbance introduced by a single mutation. A similar observation was previously made with insertions of amino acids. A single insertion of alanine in the N-terminal i3 loop disrupted signaling in the m3 receptor, whereas a double insertion resulted in normal coupling(17) .

The Arg-Ile-Tyr-Lys (RIYK) motif in the N terminus of the i3 loop, which is identical in m1 and m3 (Fig. 1), was shown to be critical to coupling to PI turnover in the rat m3 receptor(12) . When analyzing the corresponding single point mutations, only the Tyr mutation strongly reduced coupling to PI hydrolysis, whereas mutating Ile to Ala had no effect on m3-evoked PI turnover(12) . These Ile and Tyr residues are equivalent to Ile-211 and Tyr-212 of Hm1 mutated in the present study (Fig. 1), but in contrast to the results with m3, mutation I211A affected the coupling of Hm1 more strongly than Y212A. If these residues, located in a highly conserved region of the i3 N-junction, were directly involved in G protein coupling, we would have expected the same mutational effects for Hm1 and Hm3, which was not the case. If the conformation of the i3 loop junctions, rather than single amino acid side chains, is the main factor in G protein coupling that is affected by point mutations, different effects in m1 and m3 can be expected as the large middle portions of the i3 loops of Hm1 and Hm3 are strikingly different and may acquire different structural orientations upon mutation of the junctions. Hence, for proper i3 loop orientations, different amino acids may be critical in m1 and m3. However, our results do not rule out any direct interaction between this i3 domain and the G protein. Moreover, different results with m1 and m3 may also be due to use of different expression systems (Cos-7 versus HEK) possibly expressing different G proteins. Shapiro et al.(10) reported that mutations may have different effects in different cell lines, which suggests that different G proteins are involved in signal transduction.

The importance of the C-terminal region of the i3 loop to coupling is also documented in several studies (see, e.g., Refs. 3, 10, and 22). We previously found that coupling of the double mutant K359A/K361A of Hm1 was similar to that of the wild type(21) , whereas the triple mutant E360A/K362A/T366A was strongly defective (4) (Table 1). Therefore, the three corresponding single point mutations were constructed. Mutant T366A is as effective in coupling to PI turnover as the wild type Hm1 (Table 1). In contrast, K362A is highly defective; moreover, it is significantly (p leq 0.05) more defective than the triple mutant E360A/K362A/T366A even though its level of expression is higher (Table 1). Mutant K362A also displays a 5-fold lower binding affinity to the agonist (Table 2). Again, multiple mutations may have partially compensated for the effects of the single point mutation, which can be attributed to conformational compensation.

The i3 C terminus of several G protein-coupled receptors is rich in basic amino acids, which have been suggested to be required in G protein activation. Okamoto and Nishimoto (29) found that the motif of BBXB or BBXXB (B stands for a basic, X for a non-basic residue) is required for G protein activation, and such motifs are present in the C-terminal i3 loop junction of several subtypes of muscarinic, adrenergic, and dopaminergic receptors. However, activating mutations of the adrenergic receptors (24) and of Hm1 (this study) were independent of the charge of the substitution, which favors the notion that conformational changes determine receptor activity. Although mutation K362A was indeed strongly inactivating, the double m1 mutation K359A/K361A was capable of coupling to PI turnover(21) , arguing against the BBX(X)B activating motif in this case.

A very different result is obtained with mutant E360A, which shows a 10-fold enhancement in binding affinity to carbachol relative to Hm1 WT (Fig. 2, Table 1). Moreover, the carbachol EC value for stimulating PI turnover was 1.17 ± 0.89 µM for E360A (three independent dose-response curves, and approximately 1 µM in a repeat experiments with a limited low carbachol concentration range), indicating that the E360A mutation also sensitized the receptor to carbachol, relative to the wild type receptor (EC = 7.4 ± 0.1 µM) (Fig. 3). Moreover, transfection with E360A resulted in higher base-line levels of PI release compared to the wild type, for stimulating PI turnover (Table 1), while the maximal response is similar or slightly higher than the maximal response of the Hm1 WT to carbachol stimulation (Fig. 3). To determine whether the elevated base-line level correlates with the level of receptor expression, receptor density was plotted against basal PI turnover (Fig. 4). A strong relationship was observed between receptor number and basal activity for the E360A mutant, whereas the slope factor for Hm1 WT did not differ significantly from zero. Therefore, the PI turnover stimulated by E360A in the absence of agonist was significantly greater than that of the wild type.


Figure 4: Relationship between basal PI turnover (in the absence of agonist) and receptor density of wild type Hm1 and activating mutant E360A. Values of basal PI turnover stimulation were fitted to a linear function. For the activating mutant E360A basal PI turnover and receptor density were highly correlated (coefficient of correlation r = 0.94, p leq 0.001). In contrast, basal PI turnover and receptor density are not correlated for wild type Hm1. Filledcircles are for the non-transfected cells.



Significantly enhanced basal coupling activity, enhanced affinity to agonist, and enhanced agonist potency are hallmarks of an activating mutation which results in constitutive receptor activity in the absence of any agonist(32) . Constitutive receptor activity can be suppressed with an antagonist having negative intrinsic activity, but no such negative muscarinic antagonist had been described as yet. We selected atropine as an antagonist with potential negative intrinsic activity. Indeed, the elevated base-line level achieved with E360A was largely antagonized by 10 µM atropine, and two full atropine dose-response curves gave an IC value of 0.55 ± 0.09 nM atropine (Fig. 5). This result is consistent with the high affinity of atropine at muscarinic receptors, and it indicates that the negative activity of atropine is exerted at the m1 receptor, rather than at other nonspecific sites. Thus, atropine behaves as an antagonist with negative intrinsic activity, decreasing PI levels even in the absence of any agonist. Negative intrinsic activity was also described recently for 5-HT receptor antagonists(33) , and for µ-receptor antagonists(34) .


Figure 5: Atropine dose-response curve for suppressing basal PI levels of the activating mutant E360A and Hm1 wild type. Basal PI turnover in the absence of agonist was significantly higher for E360A over the wild type. Atropine concentration of 0.1 to 1000 nM were used in the E360A dose-response curves (mean of two independent experiments, each with triplicates; n = 6). Four other independent experiments using atropine dose levels of 1 nM to 10 µM) replicated these results. Expression of wild type Hm1, measured with [^3H]NMS to determine cell surface-accessible receptors, was 2400 fmol/mg protein in the experiment shown, and for E360A the expression levels were 2388 and 740 fmol/mg protein, for the two experiments. The calculated IC of atropine for E360A was 0.55 ± 0.09 nM.



These results raised the question whether basal activity was also detectable for the Hm1 wild-type receptor. Transfection of Hm1 WT into HEK 293 cells caused only minimal changes in basal PI turnover, except for experiments with high receptor expression. However, even with high expression levels, basal levels were detectably elevated only in some experiments, suggesting that variable cell conditions may play a role. Since such small changes in basal levels may in part reflect cellular adjustments in labeling IP pools with myo-[^3H]inositol, we then tested whether atropine had any effect on basal levels in Hm1 WT-transfected cells. Again, the results were variable, ranging from no measurable effect in several experiments to a very small decrease in some experiments with high receptor expression. An atropine dose-response curve with Hm1 WT (2400 fmol/mg protein) is shown in Fig. 5, in which case there was a detectable decrease of basal PI turnover at 1 µM atropine from 1.02 ± 004% to 0.85 ± 0.04% (significant at 95% confidence in the Fisher PLSD test). From these data, we conclude that any basal activity of Hm1 in HEK 293 cells is minimal, if present at all.

Constitutively active mutants involving the i3 loop C-junction had been described for the adrenergic receptors(24, 25, 26, 27) , but this is the first report of an activated mutant for the muscarinic acetylcholine receptor. Activating mutations of the intracellular receptor portion were all localized in the C terminus of the i3 loop, presumably causing a change in i3 loop conformation to activate the receptor. Multiple other activating mutations have recently been identified, distributed throughout the entire receptor protein(35, 36) , suggesting that GPCRs may require little energy for the transition from an inactive to an active state. Furthermore, activating mutations have been associated with several genetic disorders, emphasizing the physiological relevance of basal receptor activity(35, 36) . It remains to be seen to what extent basal activity of muscarinic receptors plays a physiological role, but our finding of an activating m1 mutation facilitates studies on basal muscarinic tone.

On the basis of these results, we propose the following activation scheme for G protein-coupled receptors. Upon agonist stimulation, the intracellular loops and the N-terminal tail rearrange to uncover a binding pocket that can accommodate a number of proteins responsible for downstream effects. The hinge regions of the i3 loop may be crucial to this rearrangement, exposing multiple direct coupling sites on the intracellular portions of the receptor. The proteins with access to this activation binding pocket could include G proteins, internalization and down-regulation factors, receptor kinases, and arrestins that facilitate receptor desensitization. Modification of the i3 loop configuration could selectively affect access of any of these factors to the agonist-induced binding pocket, thereby accounting for unpredictable results with i3 loop mutations. Therefore, our mutational results are insufficient to define direct receptor-G protein coupling domains which may comprise multiple sites along the cytoplasmic face of the receptor.


FOOTNOTES

*
This work was supported by National Institutes of Health NIGMS Grants GM 43102 and GM 08388 (to M. S. S.), NIMH Grant K21 MH 00996, and grants from the German Research Society (to D. F. G. and P. H.) and AFPE (to M. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 415-476-1947; Fax: 415-476-0464.

(^1)
The abbreviations used are: GPCR, G protein-coupled receptor; TMD, transmembrane domain; NMS, N-methylscopolamine; TMD, transmembrane domain; WT, wild type; PI, phosphatidylinositol.


REFERENCES

  1. Wong, S. K.-F., Parker, E. M., and Ross, E. M. (1990) J. Biol. Chem. 265, 6219-6224 [Abstract/Free Full Text]
  2. Franke, R. R., König, B., Sakmar, T. P., Khorana, H. G., and Hofmann, K. P. (1990) Science 250, 123-125 [Medline] [Order article via Infotrieve]
  3. Dalman, H. M., and Neubig, R. R. (1991) J. Biol. Chem. 266, 11025-11029 [Abstract/Free Full Text]
  4. Moro, O., Lameh, J., Högger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276 [Abstract/Free Full Text]
  5. Moro, O., Shockley, M. S., Lameh, J., and Sadée, W. (1994) J. Biol. Chem. 269, 6651-6655 [Abstract/Free Full Text]
  6. König, B., Arendt, A., Mcdowell, J. H., Kahlert, M., Hargrave, P. A., and Hofmann, K. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6878-6882 [Abstract]
  7. Liggett, S. B., Caron, M. G., Lefkowitz, R. J., and Hnatowich, M. (1991) J. Biol. Chem. 266, 4816-4821 [Abstract/Free Full Text]
  8. Wess, J., Brann, M. R., and Bonner, T. I. (1989) FEBS Lett. 258, 133-136 [CrossRef][Medline] [Order article via Infotrieve]
  9. Cotecchia, S., Ostrowski, J., Kjelsberg, M. A., Caron, M. C., and Lefkowitz, R. J. (1992) J. Biol. Chem. 267, 1633-1639 [Abstract/Free Full Text]
  10. Kunkel, M. T., and Peralta, E. G. (1993) EMBO J. 12, 3809-3815 [Abstract]
  11. Shapiro, R. A., Palmer, D., and Cislo, T. (1993) J. Biol. Chem. 268, 21734-21738 [Abstract/Free Full Text]
  12. Blüml, K., Mutschler, E., and Wess, J. (1994) J. Biol. Chem. 269, 402-405 [Abstract/Free Full Text]
  13. Wade, S. M., Dalman, H. M., Yang, S.-Z., and Neubig, R. R. (1994) J. Biol. Chem. 45, 1191-1197
  14. Maeda, S., Lameh, J., Mallet, W. G., Philip, M., Ramachandran, J., and Sadée, W. (1990) FEBS Lett. 269, 386-388 [CrossRef][Medline] [Order article via Infotrieve]
  15. Moro, O., Lameh, J., and Sadée, W. (1993) J. Biol. Chem. 268, 6862-6865 [Abstract/Free Full Text]
  16. Probst, W. C., Snyder, L. A., Schuster, D. I., Brosius, J., and Sealfon, S. C. (1992) DNA Cell Biol. 11, 1-20 [Medline] [Order article via Infotrieve]
  17. Duerson, K., Carroll, R., and Clapham, D. (1993) FEBS Lett. 324, 103-108 [CrossRef][Medline] [Order article via Infotrieve]
  18. Cheung, A. H., Huang, R.-R. C., and Strader, C. D. (1992) Mol. Pharmacol. 41, 1061-1065 [Abstract]
  19. Voss, T., Wallner, E., Czernilofsky, A. P., and Freissmuth, M. (1993) J. Biol. Chem. 268, 4637-4642 [Abstract/Free Full Text]
  20. Lechleitner, J., Hellmiss, R., Duerson, K., Ennulat, D., David, N., Clapham, D., and Peralta, E. (1990) EMBO J. 9, 4381-4390 [Abstract]
  21. Arden, J. R., Nagata, O., Shockley, M. S., Philip, M., Lameh, J., and Sadée, W. (1992) Biochem. Biophys. Res. Commun. 188, 1111-1115 [Medline] [Order article via Infotrieve]
  22. Palm, D., Münch, G., Dees, C., and Hekman, M. (1989) FEBS Lett. 254, 89-93 [CrossRef][Medline] [Order article via Infotrieve]
  23. Cheung, A. H., Huang, R.-R. C., Graziano, M. P., and Strader, C. D. (1991) FEBS Lett. 279, 277-280 [CrossRef][Medline] [Order article via Infotrieve]
  24. Kjelsberg, M. A., Cotecchia, S., Ostrowski, J., Caron, M. G., and Lefkowitz, R. J. (1992) J. Biol. Chem. 276, 1430-1433
  25. Ren, Q., Kurose, H., Lefkowitz, R. J., and Cotecchia, S. (1993) J. Biol. Chem. 268, 16483-16487 [Abstract/Free Full Text]
  26. Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 4625-4636 [Abstract/Free Full Text]
  27. Pei, G., Samama, P., Lohse, M., Wang, M., Codina, J., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2699-2702 [Abstract]
  28. Okamoto, T., Katada, T., Murayama, Y., Ui, M., Ogata, E., and Nishimoto, I. (1990) Cell 62, 709-717 [Medline] [Order article via Infotrieve]
  29. Okamoto, T., and Nishimoto, I. (1992) J. Biol. Chem. 267, 8342-8346 [Abstract/Free Full Text]
  30. Deng, W. P., and Nickoloff, J. A. (1993) Anal. Biochem. 200, 81-88 [CrossRef]
  31. Lameh, J., Philip, M., Sharma, Y. K., Moro, O., Ramachandran, J., and Sadée, W. (1992) J. Biol. Chem. 267, 13406-13412 [Abstract/Free Full Text]
  32. Tiberi, M., and Caron, M. G. (1994) J. Biol. Chem. 269, 27925-27931 [Abstract/Free Full Text]
  33. Barker, E. L., Westphal, R. S., Schmidt, D., and Sanders-Bush, E. (1994) J. Biol. Chem. 269, 11687-11690 [Abstract/Free Full Text]
  34. Wang, Z., Bilsky, E. J., Porreca, F., and Sadée, W. (1994) Life Sci. 54, 339-350
  35. Lefkowitz, R. J. (1993) Science 365, 603-604
  36. Coughlin, S. R. (1994) Curr. Opin. Cell Biol. 6, 191-197 [Medline] [Order article via Infotrieve]

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