©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure-Function of Muscarinic Receptor Coupling to G Proteins
RANDOM SATURATION MUTAGENESIS IDENTIFIES A CRITICAL DETERMINANT OF RECEPTOR AFFINITY FOR G PROTEINS (*)

(Received for publication, October 25, 1994)

Ethan S. Burstein (1)(§) Tracy A. Spalding (1) David Hill-Eubanks (1) (2) Mark R. Brann (1) (2)

From the  (1)Molecular Neuropharmacology Section, Departments of Psychiatry and Pharmacology, and Vermont Cancer Center, University of Vermont, Burlington, Vermont 05405 and (2)Receptor Technologies Inc., Winooski, Vermont 05404

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To derive structure-function relationships for receptor-G protein coupling, libraries were created of human m5 muscarinic acetylcholine receptors (m5) randomly mutated in the C-terminal region of the third intracellular loop. Functional receptors were identified based on their ability to amplify NIH 3T3 cells in a ligand-dependent manner. These receptors either had wild-type phenotypes (Group 1) or were functionally impaired (Group 2). No ``activated receptors'' were identified. Tolerated substitutions in Group 2 receptors were randomly distributed and frequently included prolines and glycines. In contrast, tolerated substitutions in Group 1 receptors exhibited a periodicity proximal to transmembrane domain 6 where proline and glycine substitutions were not observed. These observations are consistent with a short alpha-helical extension of the C-terminal region of the third intracellular loop from transmembrane domain 6. Mutations at Ala-441 were most commonly associated with impaired function of Group 2 receptors. Twelve point mutations at Ala-441 were tested, and all caused marked increases in EC values with little effect on maximal response or agonist binding affinity. These results indicate that Ala-441 is a key determinant of m5 receptor affinity for G proteins and exists within the structural context of a short alpha-helix.


INTRODUCTION

Muscarinic acetylcholine receptors are members of a large family of receptors that mediate signal transduction by coupling with G proteins. Most members of this family display significant sequence homology, particularly within regions predicted to form seven transmembrane domains (TM 1-7)(^1)(1, 2) . Muscarinic receptors consist of five genetically defined subtypes (m1-m5) (3, 4, 5, 6) that can be divided into at least two functional classes. m1, m3, and m5 couple to pertussis toxin-insensitive G proteins to potently stimulate phospholipase C, promote calcium release from cytosolic stores, modulate ion channels, stimulate mitogenesis, and transform NIH 3T3 cells. m2 and m4 couple to pertussis toxin-sensitive G proteins to weakly stimulate phospholipase C, open inwardly rectifying potassium channels, and inhibit adenylyl cyclase. The m2 and m4 subtypes do not stimulate mitogenesis nor do they transform NIH 3T3 cells(7, 8) . By preparing chimeras between subtypes from the two functional classes (e.g. chimeric m1/m2 and m3/m2 receptors), the third cytoplasmic loop (i3) has been shown to be the region that defines subtype specificity for distinct G proteins (9, 10, 11, 12) . The central portion of the i3 loop can be deleted without impairing coupling to G proteins, indicating that only the N- and C-terminal regions of the i3 loop (Ni3 and Ci3, adjacent to TM5 and TM6, respectively) are required for function(13, 14, 15, 16) . However, the precise structural requirements of these domains for G protein coupling remain largely unknown.

In order to comprehensively examine the structure-function relationships of receptor-G protein coupling, we have developed an efficient method for using random mutagenesis in mammalian systems. A major advantage of random mutagenesis is the ability to create thousands of amino acid substitutions with no bias regarding the type and location of substitutions made. By using a screen to identify mutants that retain function, one can identify which amino acid substitutions are functionally allowed. Activated phenotypes or ``gain of function mutations'' are particularly easy to identify in a screen based upon positive selection. When a domain is saturated with mutations, one can make inferences about amino acid substitutions that are never recovered as functional proteins(18, 19) .

A reliable and facile screen is a prerequisite for any random mutagenesis protocol. Our methods are based on the observation that phosphatidylinositol-coupled muscarinic receptors induce ligand-dependent focus formation in NIH 3T3 cells. In fact, the dose-response relationships of transformation and phospholipase C stimulation are identical(28) . Thus, functional muscarinic receptors can be cloned from a library of mutants by virtue of their ability to induce foci in response to agonist treatment. Furthermore, we have found that this receptor-mediated stimulatory effect can be monitored using a reporter gene assay, (^2)eliminating the need to culture cells until foci develop and allowing us to measure graded responses. We have used these methodologies to obtain the first comprehensive look at the structurally important elements of the i3 loop of a G protein-coupled receptor.


EXPERIMENTAL PROCEDURES

Library Construction

The m5 receptor was subjected to mutagenesis over the C-terminal 22 amino acids of the third intracellular loop (Ci3), adjacent to TM6. Two PCR products were prepared such that the reverse primer (P2) for the first product comprised the entire Ci3 domain and the forward primer (P3) for the second product comprised the entire TM6 domain. To incorporate mutations, an equimolar mixture of the four bases was substituted at a 15% rate for wild-type nucleotides during synthesis of the P2 primer. This approach allows quantitative control of the rate of base misincorporation and ensures a completely random distribution of mutations. This doping rate was chosen to ensure that relatively few unaltered receptors are selected and that a significant proportion of recombinants is functional. At this mutation rate, there is less than a 2% chance that any residue within Ci3 will not be mutated at least once in any 20 clones selected at random. The outer primers (P1 and P4) contain ApaI and XbaI restriction sites for subsequent cloning. The two PCR products were treated with T4 DNA polymerase to create blunt ends, ligated to yield concatamers, and restricted with ApaI and XbaI to release the randomly mutated (*) Ci3*ApaI/XbaI inserts. Inserts were ligated into a ApaI/XbaI fragment of the pcD-m5 yielding a population of mutant m5 receptor DNA (pCD-m5-Ci3*). Competent Escherichia coli were transformed with pCDm5-Ci3* and amplified either as pools or as individuals. Plasmid DNA was isolated and used for sequencing and functional assays.

Cell Culture

NIH 3T3 cells (ATCC no. CRL 1658) and COS7 cells were incubated at 37 °C in a humidified atmosphere (5% CO(2)) in Dulbecco's modified Eagle's medium supplemented with 4500 mg/liter glucose, 4 nML-glutamine, 50 units/ml penicillin G, 50 units/ml streptomycin (A.B.I.), and 10% calf serum for 3T3 cells or 10% fetal bovine serum for COS7 cells (Life Technologies, Inc.).

Transfection Procedure and Functional Assays

Receptor Selection and Amplification Technology (R-SAT) assays were performed as follows. Cells were plated 1 day before transfection using 1 times 10^6 cells in 10 ml of media/10-cm plate. Cells were transfected by calcium precipitation as described by Wigler et al.(29) , using 5 µg of the human m5 receptor (6) in the pcD expression vector(33) , 5 µg of pSV-beta-galactosidase (Promega, Madison, WI), and 20 µg of salmon sperm DNA (Sigma). One day after transfection the medium was changed, and after 2 days cells were trypsinized, and aliquots were placed in the wells of a 96-well plate (100 µl/well). One 10-cm plate yields enough cells for 96 wells. Ligands were combined with the cells to a final volume of 200 µl/well. After 5 days in culture (except for time course studies) beta-galactosidase levels were measured essentially as described by Lim and Chae(30) . The medium was aspirated from the wells, and the cells were rinsed with phosphate-buffered saline (136.9 mM NaCl, 2.68 mM KCl, 8.09 mM Na(2)HPO(4), and 1.47 mM KH(2)PO(4), pH 7.4). 200 µl of phosphate-buffered saline with 3.5 mMo-nitrophenyl beta-D-galactopyranoside and 0.5% Nonidet P-40 (Sigma) was added to each well, and the 96-well plate was incubated at room temperature. After 16 h the plates were read at 405 nm on a plate reader (Bio-Tek EL 310 or Molecular Devices). Data from R-SAT assays were fit to the equation, R = D + (A - D)/(1 + (x/c)), where A is minimum response, D is maximum response, and c is EC (R is response and x is concentration of ligand). Curves were generated by least-squares fits using the program KaleidaGraph (Abelbeck Software). Focus assays were performed as described(28) .

Binding Studies

For all binding studies, receptors were transiently expressed in COS7 cells, and cells were harvested 72 h after transfection and stored at -80 °C. Membranes were prepared in binding buffer containing 25 mM sodium phosphate (pH 7.4) and 5 mM magnesium as described (31) immediately before use. Binding reactions were allowed to proceed at room temperature for 4 h. Reactions were terminated by filtration. Filters were washed with ice-cold buffer and dried, and bound radioactivity was counted. Nonspecific binding was assessed in the presence of 1 µM atropine(31) . The dissociation constant (K(d)) for carbachol was calculated by the method of Cheng and Prusoff (17) using IC values assessed in the presence of 400 pM [^3H]N-methylscopolamine (NMS) as described (31) . Data from binding experiments were determined to fit mass action relationships and were fit to a = (B(max)x/K(d))/(1 + x/K(d)), where a is [^3H]NMS specifically bound and x is [^3H]NMS concentration, to obtain the dissociation constant K(d) and the total number of binding sites B(max).


RESULTS

Based on protocols that we have previously used to evaluate induction of foci by ligand-occupied muscarinic receptors(28) , we developed a method for monitoring proliferative responses using a reporter gene. In our assay technology, ligands select and amplify cells that express functional receptors. These same cells also express a reporter gene; thus receptor activity is assayed as a change in enzyme levels (R-SAT, patent pending). As shown in Fig. 1A, when beta-galactosidase and m5 muscarinic acetylcholine receptor expression vectors were co-transfected into NIH 3T3 cells, there was a time-dependent amplification of cells cultured in the presence of carbachol compared with cells cultured in media alone (Fig. 1). This amplification was rapid, becoming significant by the second day of culture and plateauing by 5 days in culture. Thereafter all assays were performed for 5 days. We then evaluated the dose-response relationship of carbachol-induced amplification and compared it with a focus-forming assay. As shown in Fig. 1B, the EC for carbachol was approximately 10-fold lower in R-SAT than focus assays. This difference is probably due to higher expression levels of receptor in the shorter assay (5 days compared with 14 days). The pharmacology of carbachol and several other muscarinic agonists and antagonists as determined with R-SAT is very similar to that determined with traditional functional assays. A detailed pharmacological analysis will be presented elsewhere.^2


Figure 1: R-SAT assay. A, time course. m5 and beta-galactosidase cDNAs were co-transfected into NIH 3T3 cells, and induced beta-galactosidase activity was assayed at the indicated times as described under ``Experimental Procedures.'' The responses were calculated from the base line, and maximum responses were derived from least squares fits of full dose-responses at each time point (filled triangles, maximal response; open circles, basal response). B, carbachol dose-response of the beta-galactosidase amplification on day 5 and focus formation on day 14. Plotted are absorbance at 405 nm (filled triangles, R-SAT) and number of macroscopic foci/10-cm^2 dish (open squares, focus assay) versus carbachol concentration. Assays and curve fits were performed as described under ``Experimental Procedures.''



We employed R-SAT in combination with random saturation mutagenesis to investigate the structure-function relationships of m5 receptor-G protein coupling. We used randomly doped oligonucleotides in a PCR-based construction method (Fig. 2) to generate a library of mutant m5 muscarinic acetylcholine receptors saturated with mutations throughout Ci3. Sequence analysis of unscreened libraries verified that there was a 10% nucleotide substitution rate with an average of greater than 5 amino acid substitutions per clone (not shown). A pool of recombinants representing 500 clones was evaluated for the ability to bind the antagonist NMS and to transform NIH 3T3 cells. We observed that transient expression of pCDm5-Ci3* in COS7 cells yielded 35% of the binding sites compared with cells transfected with an equivalent amount of wild-type pCDm5 (1.1 ± 0.2 versus 3.1 ± 0.3 pmol/mg protein) with essentially identical affinity for NMS (153 ± 26 pMversus 126 ± 19 pM), indicating that a third of the constructs expressed binding sites. The Ci3* pool of mutants was able to transform 3T3 cells 5-10% as efficiently as wild-type m5 (not shown), consistent with the notion that there was a strong functional selection against mutations within Ci3. Interestingly, although mutations within the analogous regions of other receptors are activating(20, 21, 22, 23, 24, 25, 26) , we detected no foci in the absence of carbachol.


Figure 2: Mutagenesis of the Ci3 of m5. A, region to be mutagenized. Wild-type sequence is indicated. B, library construction strategy. PCR was performed using primers p1-p4. The p2 primer comprised residues 424-445. The outer primers (p1 and p4) contain ApaI and XbaI restriction sites for subsequent cloning. The two PCR products were treated with T4 DNA polymerase to create blunt ends, ligated to yield concatamers, and restricted with ApaI and XbaI to release the randomly mutated (*) Ci3*ApaI-XbaI inserts. Inserts were ligated into an ApaI/XbaI fragment of the pcD-m5 yielding a population of mutant m5 receptor DNA (pCDm5-Ci3*). Competent DH5alpha cells were transformed with pCDm5-Ci3*, amplified either as pools or as individual transformants, and plasmid DNA was isolated.



The results in the focus experiments with the pooled library indicated that there was a sufficiently high percentage of functional mutants to screen clones derived from this library individually, eliminating the need to retrospectively clone and identify positive receptors. Assuming that mutant receptors capable of producing foci in 3T3 cells would also be able to mount a response in the R-SAT assays, we expected a 5-10% positive clone rate. We screened 300 individual constructs in the presence or absence of a single dose (100 µM) of carbachol in triplicate and were able to identify 36 clones that responded. Three of these clones failed to respond a second time, and two clones had wild-type sequences.

The positive clones identified in the primary screen were subjected to a detailed dose-response analysis, using doses of carbachol ranging from 1 nM to 100 µM or 100-fold below to 1000-fold above the EC of carbachol for wild-type m5. As shown in Fig. 3and Table 1, we isolated receptors with EC values ranging to 100-fold above the wild-type EC and maximal responses ranging from 25 to 125% of wild type. Thus we effectively isolated the full range of receptor phenotypes with rightward shifts in EC or with decreased maximum responses that we could have reliably identified under these conditions (the EC cannot be greater than the K(d)). However, we did not find any receptors with greater than 3-fold leftward shifts in EC or significantly elevated activity. We would have expected, a priori, that activated receptors would have been readily detected since R-SAT is based upon positive selection and since mutations in Ci3 activate many other receptors(20, 21, 22, 23, 24, 25, 26) .


Figure 3: Dose-response curves in the R-SAT functional assay for Group 1 (fully functional receptors) (A) and Group 2 (functionally impaired receptors) (B) compared with m5.





We divided the functional mutant receptors into two groups based upon their pharmacological phenotypes ( Table 1and Fig. 3). Group 1 includes receptors with essentially wild-type characteristics, whereas Group 2 receptors are impaired but still functionally competent. To assess the molecular basis for the range of phenotypes observed, we sequenced each of these receptors. Examination of the receptors in Group 1 reveals that although mutations were randomly introduced, functionally tolerated mutations were clustered in residues 424-438 and rare in residues 439-445, suggesting there was functional selection against mutations at discrete residues. Indeed, there was an average of 3.7 substitutions per Group 1 receptor but 4.9 substitutions per Group 2 receptor supporting this notion.

To facilitate comparison of the molecular differences between Group 1 and Group 2 receptors, we compiled all the mutations into lists (Table 2). A consensus sequence of functionally conserved residues that were never mutated can be derived for the Group 1 receptors consisting of Ala-440, Ala-441, and Leu-444 (Table 2). In addition, Lys-439 was only conservatively mutated to arginine. Group 2 receptors had a much more random distribution of mutations, and 9 out of 16 had mutations in the consensus sequence derived for Group 1, confirming the functional importance of these residues. Merely examining sequence conservation among muscarinic subtypes (Table 2) was not reliably predictive of the functional importance of any specific residue. For example, the KERK motif (amino acids 436-439) is well conserved, but only the second lysine appeared to have functional importance based on our analysis (Table 2), which is consistent with earlier observations(15, 16) . We assumed residues observed to be frequently or radically mutated in receptors that retained full function or were dispensable for function. Presumably, mutations observed in both groups also did not account for the observed phenotypic changes in Group 2 nor could differences in expression levels account for the relative impairment of Group 2 receptors compared with Group 1 receptors (see [^3H]NMS binding in the legend to Table 1). Thus the impaired function of Group 2 receptors is most likely related to the mutations within the consensus sequence.



Another striking difference between Group 1 and Group 2 receptors is the proline and glycine substitution patterns (Table 2, see helix breaks). Proline and glycine substitutions are excluded from residues 439-445 in Group 1 receptors but are distributed throughout Group 2 receptors, including residues 439-445. This is strong evidence for the existence of a short alpha-helical extension from TM6, which when disrupted impairs receptor function. It is noteworthy that the mutation pattern in Group 1 receptors appears to become periodic beginning at Arg-438 with radical mutations clustered 4 residues apart (Arg-438-Gln-442), also suggesting the existence of a helical structure. The junction of the i3 loop and TM6 is predicted to be within the vicinity of Gln-442(8) . Together, these data suggest that the TM6 helix may extend into Ci3 for a single turn before adopting some other conformation.

Inspection of the Group 2 mutants revealed that mutations at Ala-441 were associated with large increases in EC (see clones 32, 135, 139, 166, and 202 in Table 1), implying that Ala-441 is a critical determinant of receptor potency. This hypothesis is complicated since the mutations at Ala-441 were analyzed in the context of other mutations. Therefore, we constructed a point mutant library of receptors randomly mutated to all of the possible amino acid substitutions at Ala-441, rescued viable clones by R-SAT, and analyzed each receptor as described above. We identified 12 unique amino acid substitutions at Ala-441, spanning all of the major amino acid classes, and each caused significant increases in EC without affecting maximal response (Table 3). Changes in agonist binding affinity could not account for the increases in EC nor could differences in expression levels (Table 3, legend). Significantly, substitution of threonine, which is found in the analogous position to Ala-441 in m2 and m4 (G(i)-coupled) receptors (see Table 2and (8) ) caused the largest shift in EC suggesting Ala-441 also influences G protein coupling selectivity. Proline, which disrupts alpha-helices, was also effective at reducing receptor potency. Cysteine had little effect, perhaps because it is of similar size to alanine. However basic, acidic, aromatic, and large hydrophobic residues all had similar effects on function.




DISCUSSION

We have developed an efficient method for analysis of the structure-function relationships of G protein-coupled receptors by random mutagenesis. This approach was possible because of the development of a high throughput assay based upon cellular proliferation, which offers significant technical advantages over focus assays or second messenger assays for evaluating receptor function. Second messenger assays are too labor intensive to allow one to screen the large numbers of receptors necessary for studying structure-function relationships by random mutagenesis. Although focus assays are amenable to screening large numbers of clones, foci are not observed until at least 10 days of carbachol treatment(28) , while R-SAT gives measurable responses in 3-5 days. More significantly, one can easily measure graded responses using R-SAT, allowing one to characterize subtle effects of mutations upon receptor function, whereas focus assays only discriminate all or none responses.

Using R-SAT in combination with random saturation mutagenesis we have discovered that the section of Ci3 proximal to TM6 regulates m5 receptor affinity for G proteins and that this function is most severely impaired by proline substitutions or by substitutions at Ala-441 (Fig. 4). Therefore we conclude that Ala-441 is a key determinant of m5 affinity for G proteins and that Ala-441 is likely to reside within the structural context of a short alpha-helical extension from TM6. We have found that overexpression of G(q) shifts the EC values of ``low potency'' receptors (EC > 1 µM) back to wild-type values, (^3)further supporting the idea that the main effect of mutations at Ala-441 is to reduce receptor affinity for G(q), although it is impossible to say whether or not Ci3 directly contacts G(q).


Figure 4: Ratio of EC values of each clone to the EC of wild-type m5. Inset, plot for the Ala-441 point mutants. A value below 1 represents a clone with greater potency than wild-type m5. EC values were determined from R-SAT experiments performed as described under ``Experimental Procedures.'' P and D denote proline substitutions or deletions observed within the predicted helical segment of Ci3, respectively. Three-letter amino acids denote observed substitutions for Ala-440. Group 1 and Group 2 clones are defined as in Fig. 2and Table 1.



We could detect no evidence of elevated constitutive activity of any of the Ci3 mutants in contrast to results for many other receptors (20, 21, 22, 23, 24, 25, 26) . Similarly, none of the Ala-441 point mutants were activated, in contrast to studies on the alpha-adrenergic receptor in which all amino acid substitutions at the analogous position activated that receptor(22) . These negative results are not due to technical limitations since the activated phenotypes of mutationally activated adrenergic receptors are readily observed using R-SAT. (^4)Although we found no activating mutations within Ci3, we have discovered that m5 is activated by mutations in TM6. (^5)TM6 is also a site of activation for the leutinizing hormone receptor(32) . Mutations in TM6 increase the basal activity of m5 and also raise the affinity of m5 for agonists, very similar to the effects of mutations within the Ci3 region of the adrenergic receptors(20, 21) . Considering that TM6 is immediately adjacent to Ci3 and is predicted to form an alpha-helix extending into Ci3 (this paper) one might envision TM6-Ci3 as a single functional unit extending down to Lys-439. In this context, the function of the TM6-Ci3 domain of m5 may be quite similar to the adrenergic receptors, but the details of how it operates to convert agonist binding into G protein coupling may be reversed in the two receptor families.


FOOTNOTES

*
This work was supported in part by Eli Lilly Research Laboratories, Allergan Pharmaceuticals, Schering Plough Pharmaceuticals, and Vermont Experimental Program to Stimulate Competitive Research. 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.

§
Supported by National Research Service Award fellowship F32NS09436. To whom correspondence should be addressed.

(^1)
The abbreviations used are: TM, transmembrane domain; PCR, polymerase chain reaction; R-SAT, receptor-selection and amplification technology; NMS, N-methylscopolamine; Ci3, C-terminal region of the i3 loop.

(^2)
H. B. Jørgensen and M. R. Brann, submitted for publication.

(^3)
E. S. Burstein, T. A. Spalding, D. Hill-Eubanks, and M. R. Brann, unpublished observations.

(^4)
D. Hill-Eubanks, E. S. Burstein, and M. R. Brann, unpublished observations.

(^5)
T. A. Spalding, E. S. Burstein, H. B. Jørgensen, D. Hill-Eubanks, and M. R. Brann, submitted for publication.


ACKNOWLEDGEMENTS

We thank R. Lefkowitz for the generous gift of activated alpha- and alpha-adrenergic receptors, B. Conklin for GalphaQ, and I. Macara for critical reading of the manuscript.


REFERENCES

  1. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688 [CrossRef][Medline] [Order article via Infotrieve]
  2. Brann, M. R. (ed) (1992) Molecular Biology of G-Protein-coupled Receptors , Birkhaeuser Boston, Inc., Cambridge, MA
  3. Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H., Kirose, T., and Numa, S. (1986) Nature 323, 411-416 [Medline] [Order article via Infotrieve]
  4. Peralta, E. G., Winslow, J. W., Peterson, G. L., Smith, D. H., Ashkenazi, A., Ramachandran, J., Schimerlink, M. I., and Capon, D. J. (1987) Science 236, 600-605 [Medline] [Order article via Infotrieve]
  5. Bonner, T. I., Buckley, N. J., Young, A. C., and Brann, M. R. (1987) Science 237, 527-532 [Medline] [Order article via Infotrieve]
  6. Bonner, T. I., Young, A. C., Brann, M. R., and Buckley, N. J. (1988) Neuron 1, 403-410 [Medline] [Order article via Infotrieve]
  7. Jones, S. V. P., Levey, A. I., Weiner, D. M., Ellis, J., Novotny, E., Yu, S., Dorje, F., Wess, J., and Brann, M. R. (1992) in Molecular Biology of G Protein-coupled Receptors (Brann, M. R., ed) pp. 170-197, Birkhaeuser Boston, Inc., Cambridge, MA
  8. Hulme, E. C., Birdsall, N. J. M., and Buckley, N. J. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 633-673 [CrossRef][Medline] [Order article via Infotrieve]
  9. Kubo, T., Bujo, H., Akiba, I., Nakai, J., Mishina, M., and Numa, S. (1988) FEBS Lett. 241, 119-125 [CrossRef][Medline] [Order article via Infotrieve]
  10. Wess, J., Brann, M. R., and Bonner, T. I. (1989) FEBS Lett. 258, 133-136 [CrossRef][Medline] [Order article via Infotrieve]
  11. Wess, J., Bonner, T. I., and Brann, M. R. (1990) Mol. Pharmacol. 38, 517-523 [Abstract]
  12. Lechleiter, J., Hellmiss, R., Duerson, K., Ennulat, D., David, N., Clapham, D., and Peralta, E. (1991) EMBO J. 9, 4381-4390 [Abstract]
  13. Shapiro, R. A., and Nathanson, N. M. (1989) Biochemistry 28, 8946-8950 [Medline] [Order article via Infotrieve]
  14. Lemeh, J., Philip, M., Sharma, Y. K., Moro, O., Ramachandran, J., and Sadee, W. (1992) J. Biol. Chem. 267, 13406-13412 [Abstract/Free Full Text]
  15. Arden, J. R., Nagata, O., Shockley, M. S., Philip, M., Lemeh, J., and Sadee, W. (1992) Biochem. Biophys. Res. Commun. 188, 1111-1115 [Medline] [Order article via Infotrieve]
  16. Kunkel, M. T., and Peralta, E. G. (1993) EMBO J. 12, 3809-3815 [Abstract]
  17. Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108 [CrossRef][Medline] [Order article via Infotrieve]
  18. Yaghmai, R., and Hazelbauer, G. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7890-7894 [Abstract]
  19. Oliphant, A. R., and Struhl, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9094-9097 [Abstract]
  20. Cotecchia, S., Exum, S. T., Caron, M. G., and Lefkowitz, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2896-2900 [Abstract]
  21. Allen, L. F., Lefkowitz, R. J., Caron, M. G., and Cotecchia, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11354-11358 [Abstract]
  22. Kjelsberg, M. A., Cotecchia, S., Ostrowski, J., Caron, M. G., and Lefkowitz, R. J. (1992) J. Biol. Chem. 267, 1430-1433 [Abstract/Free Full Text]
  23. Ren, Q., Kurose, H., Lefkowitz, R. J., and Cotecchia, S. (1993) J. Biol. Chem. 268, 16483-16487 [Abstract/Free Full Text]
  24. Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 4625-4636 [Abstract/Free Full Text]
  25. Parma, J., Duprez, L., VanSande, J., Cochaux, P., Gervy, C., Mockel, J., Dumont, J., and Vassart, G. (1993) Nature 365, 649-654 [CrossRef][Medline] [Order article via Infotrieve]
  26. Kosugi, S., Okajima, F., Ban, T., Hidaka, A., Shenker, A., and Kohn, L. D. (1993) Mol. Endocrinol. 7, 1009-1020 [Abstract]
  27. Deleted in proof
  28. Gutkind, J. S., Novotny, E., Brann, M. R., and Robbins, K. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4703-4707 [Abstract]
  29. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y., and Axel, R. (1977) Cell 11, 223-232 [Medline] [Order article via Infotrieve]
  30. Lim, K., and Chae, C.-B. (1989) BioTechniques 7, 576-579 [Medline] [Order article via Infotrieve]
  31. Wess, J., Gdula, D., and Brann, M. R. (1991) EMBO J. 10, 3729-3734 [Abstract]
  32. Shenker, A., Laue, L., Kosugi, S., Merendino, J. J., Minegishi, T., and Cutler, G. B. (1993) Nature 365, 652-654 [CrossRef][Medline] [Order article via Infotrieve]
  33. Okayama, H., and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289 [Medline] [Order article via Infotrieve]

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