©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carboxyl-terminal Domains in the Avian -Adrenergic Receptor That Regulate Agonist-promoted Endocytosis (*)

(Received for publication, November 28, 1994; and in revised form, January 10, 1995)

Eric M. Parker (§) Philip Swigart Mary H. Nunnally (¶) John P. Perkins Elliott M. Ross (**)

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Most G protein-coupled receptors, including the mammalian beta(2)-adrenergic receptor, are endocytosed to an intracellular, vesicular compartment upon continued exposure to agonist. The long form of the avian beta(1)-adrenergic receptor, which contains a carboxyl-terminal 59-amino acid extension, does not undergo agonist-promoted endocytosis. We constructed and expressed turkey beta(1)-adrenergic receptor cDNAs with regularly spaced carboxyl-terminal truncations and studied their agonist-promoted endocytosis. Removal of 34-86 amino acids from the carboxyl terminus of the turkey receptor allowed its efficient endocytosis, with optimal endocytosis observed upon removal of 59 residues. Removal of only 18 residues allowed some endocytosis. A receptor that lacks the entire carboxyl-terminal region (124 residues) was not endocytosed. We also constructed a chimeric hamster beta(2)-adrenergic receptor with the added 59-residue carboxyl-terminal domain of the turkey receptor. The chimera was not significantly endocytosed. These data indicate that residues 450-465 in the carboxyl-terminal region of the beta(1)-adrenergic receptor can act independently to block agonist-promoted endocytosis and that other carboxyl-terminal structures nearer to the seventh membrane span are required for endocytosis.


INTRODUCTION

Upon activation by agonists, G protein-coupled receptors regulate the activity of membrane-bound enzymes, ion channels, and transport proteins by catalyzing the activation of heterotrimeric G proteins ( (1) for review). Prolonged agonist binding also leads to desensitization, a decrease in the magnitude of the response despite the continued presence of agonist. At least three mechanistically and temporally distinct processes contribute to the overall phenomenon of desensitization (see (2) and (3) for review). First, agonist binding induces a rapid uncoupling of receptor from its G protein target. This process involves receptor phosphorylation by one or more protein kinases followed by binding of an inhibitory protein of the arrestin family. Second, agonist binding causes translocation of receptors from the cell surface to an intracellular vesicular compartment, probably endosomes(4, 5) . Such agonist-promoted endocytosis, also referred to in the literature as internalization or sequestration, requires somewhat longer exposure of the receptor to agonist than does the initial uncoupling reaction. Endocytosis has been proposed to play a primary role in the resensitization of receptors(6) . Finally, long term exposure of a receptor to agonist leads to down-regulation, the net loss of receptors from the cell. The mechanism of down-regulation is unclear but probably involves both degradation of endocytosed receptors and decreased receptor synthesis caused in part by destabilization of receptor mRNA(5, 7, 8, 9) .

The beta-adrenergic receptors are the most thoroughly studied members of the G protein-coupled receptor family and have been prototypes in studies of desensitization. There are three distinct subtypes of the beta-adrenergic receptor (designated beta(1), beta(2), and beta(3)(10) ). (^1)Although these isoforms are quite similar in primary structures, specificities for ligands and selectivities among G proteins, they display markedly different desensitization phenotypes. Upon exposure to agonist, the beta(2)-adrenergic receptor is uncoupled, internalized, and down-regulated(2, 3) . In contrast, the beta(3)-adrenergic receptor does not undergo any of these modes of desensitization when exposed to agonists(11) . The desensitization behavior of the beta(1)-adrenergic receptor remains unclear. Although there is general agreement that beta(1)-adrenergic receptors undergo uncoupling in response to agonist, their endocytosis and down-regulation remain controversial. In tissues, some investigators have observed down-regulation of beta(1)-adrenergic receptors and others have not, but results have generally not been confirmed for identical tissues and species in multiple laboratories (see (12) for review). Studies of cultured mammalian cells that express endogenous beta(1)-adrenergic receptors indicate that these receptors undergo all three desensitization reactions(13, 14) . However, recent studies of recombinant human beta(1)-adrenergic receptors expressed in CHW cells found desensitization but no endocytosis or degradation of receptors(15) . The turkey erythrocyte beta(1)-adrenergic receptor also does not display either endocytosis or down-regulation in erythrocytes, reticulocytes, or stably transfected L cells(16, 17, 18) .

Several lines of evidence indicate that the cytoplasmic carboxyl-terminal domain of the beta-adrenergic receptors at least partially determines their different desensitization phenotypes(6, 11, 18, 19, 20, 21, 22, 23, 24) . Hertel et al.(18) recently found that a spontaneously mutated avian beta(1)-adrenergic receptor, in which 71 carboxyl-terminal amino acid residues were lost, acquired the ability to undergo both agonist-promoted endocytosis and down-regulation. In this study, we have examined the role of the carboxyl-terminal domain of the avian beta-adrenergic receptor in modulating its endocytosis and defined separate sequences that permit and block endocytosis and shown that the endocytosis-blocking region can function when attached to other receptors.


EXPERIMENTAL PROCEDURES

Materials

Dulbecco's modified Eagle's medium (low glucose) and G418 were purchased from Life Technologies, Inc.; fetal calf serum, (-)-propranolol, and(-)-isoproterenol from Sigma; and ([I]ICYP) (^2)from DuPont NEN. CGP12177 ((-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one) was a gift from Dr. M. Staehelin (Ciba Geigy AG, Basel, Switzerland). The expression vector pCMV5 (25) was a gift from Dr. David Russell (University of Texas Southwestern Medical Center) and the cDNA encoding the hamster beta(2)-adrenergic receptor (26) was obtained from Dr. Robert Lefkowitz (Duke University). Enzymes used for molecular biology procedures were obtained from New England Biolabs.

Construction of Mutant beta-Adrenergic Receptors

cDNA for a chimeric hamster beta(2)-adrenergic receptor with the extreme carboxyl-terminal 59 amino acid residues of the turkey beta(1)-adrenergic receptor (27) was constructed by first introducing XhoI and BamHI restriction sites at the end of the hamster cDNA by site-directed mutagenesis(40) . The product was digested with XhoI and BamHI, and an XhoI-BamHI fragment of the turkey receptor cDNA was inserted.

The construction of the other truncated beta(1)-adrenergic receptor cDNAs used in this study was described previously(28) . All receptor cDNAs were inserted into the expression vector pCMV5, and the integrity of the constructs was confirmed by DNA sequencing and restriction mapping. All manipulations of recombinant DNA followed standard procedures(29) .

Expression of beta-Adrenergic Receptors in L Cells

Murine L cells (obtained from Dr. Neil Birnberg, Yale University) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal calf serum in an atmosphere of 92% air, 8% CO(2) at 37 °C. Twenty-four h prior to transfection, 5 times 10^5 cells were plated in 100-mm dishes. Cells were co-transfected with receptor expression plasmids (20 µg/dish) and the selection marker pRSVneo (1 µg/dish) by the calcium phosphate precipitation method(30) . G418-resistant cells were selected by growing the transfected cells in media supplemented with 500 µg/ml G418. Individual drug-resistant colonies were isolated after about 2 weeks and maintained in medium supplemented with 150 µg/ml G418.

Radioligand Binding Assays

The binding of [I]ICYP to beta-adrenergic receptors in L cell lysates was measured as described previously(18) . Photoaffinity labeling of receptors with [I]ICYP-diazirine was performed as described previously(28) .

Measurement of Receptor Internalization

For all internalization experiments, cells were seeded at approximately 20,000 cells/cm^2 and grown for 4 days. Cells were fed with fresh medium 24 h before each experiment. Agonist-induced internalization was then measured in two ways (see (18) for further details).

Competition with CGP12177

Cells were treated with 1 µM(-)-isoproterenol or vehicle (0.1 mM ascorbic acid) for 20 min at 37 °C. Lysates from control or isoproterenol-treated cells were assayed for CGP12177 binding according to competition with a fixed concentration of [I]ICYP. Internalized receptors are relatively insensitive to CGP12177(31, 32) , and, therefore, an increase in [I]ICYP binding in the presence of CGP12177 provides an estimate of internalized receptors (33) .

Sucrose Density Gradient Centrifugation

Hypotonic lysates from control or isoproterenol-treated cells were prepared and layered on sucrose step gradients (15%, 30%, 33%, 60% sucrose, w/v, in 20 mM Tris-Cl, pH 7.4). Gradients were centrifuged for 60 min at 28,000 rpm in a Beckman SW40Ti rotor. Fractions (0.8 ml) were collected and diluted with an equal volume of 154 mM NaCl, 5 mM MgCl(2), 20 mM Tris-Cl, pH 7.4. Receptor was measured by [I]ICYP binding as described previously, using 10 µM propranolol to define nonspecific binding(18) . Internalized receptors migrated preferentially at the 15-30% interface (low density peak) and plasma membrane-associated receptors migrated at the 33-60% interface (high density peak). The number of receptors in endocytic vesicles was assayed as specific [I]ICYP binding in the low density peak that was not inhibited by 1 µM CGP12177, and the number of the receptors in the plasma membrane fraction was assayed as specific [I]ICYP binding in the high density peak that was inhibited by CGP12177.


RESULTS

A series of mutant turkey beta(1)-adrenergic receptor cDNAs was constructed in which stop codons were inserted at evenly spaced intervals throughout the region that encodes the carboxyl-terminal domain (28) (Fig. 1). These receptors are referred to as T359, T397, T424, T449, and T465 according to the numbering of their carboxyl-terminal residues. Each truncated receptor was expressed stably in L cells, which do not have endogenous beta-adrenergic receptors. We also expressed a chimeric receptor (HTCR) in which the carboxyl-terminal 59 amino acid residues of the turkey beta(1)-adrenergic receptor were appended to the end of the hamster beta(2)-adrenergic receptor. Several clonal cell lines expressing each receptor were isolated and used in these experiments. The level of expression of the receptors in the L cell clones ranged from 90 to 850 fmol/mg of membrane protein. In general, truncated receptors and the hamster beta(2)-adrenergic receptor were expressed at higher levels than were the wild-type turkey beta(1)-adrenergic receptor or the HTCR chimera. Each mutant receptor displayed the expected size according to photoaffinity labeling with []ICYP-diazirine followed by dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography (not shown; see (28) ). When expressed in L cells, each of the receptors used here mediated activation of adenylyl cyclase by isoproterenol, as described previously for these and other constructs (see Table 2, below, and (18) ).


Figure 1: Structures of the beta-adrenergic receptors used in this study. The orientation of structural domains of the long form (encoded by spliced mRNA(34) ) of the turkey beta(1)-adrenergic receptor is shown with reference to the plasma membrane. The original and novel carboxyl termini are shown as black circles; other residues are shown as open circles. The mammalian beta(2)-adrenergic receptor and the short form of the turkey beta(1)-adrenergic receptor both terminate at a position homologous to residue 424.





The ability of the various receptors to undergo agonist-induced internalization was initially examined by determining the potency with which the hydrophilic ligand CGP12177 competes with [I]ICYP for binding to receptors in lysates prepared either from untreated cells or from cells incubated with isoproterenol for 20 min. Sequestration in endosomes, which are relatively inaccessible to hydrophilic ligands, decreases the potency of inhibition of binding by CGP12177(31, 32, 33) . As shown previously and in Fig. 2, treatment with isoproterenol increases the IC for CGP12177 binding to beta(2)-adrenergic receptors by about 15-fold. Exposure to isoproterenol does not alter the IC for the turkey beta(1)-adrenergic receptor, which is not endocytosed or down-regulated(16, 28) .


Figure 2: Effect of isoproterenol treatment on the sensitivity of beta-adrenergic receptors to CGP12177. Untreated cells (bullet) or cells that were exposed to isoproterenol for 20 min (down triangle) were lysed, and [I]ICYP binding was determined at increasing concentrations of CGP12177 as described under ``Experimental Procedures.'' Results are expressed as the percentages of maximal binding. Each panel shows a single experiment carried out in triplicate and is representative of at least 3 similar experiments (see Table 1).





In contrast to the wild-type avian beta(1)-adrenergic receptor, most of the truncation mutants were significantly endocytosed, as monitored by increases in the IC for CGP12177 ( Fig. 2and Table 1). For T424, whose carboxyl-terminal region is equivalent in length to that of the beta(2)-adrenergic receptor, isoproterenol was as effective in promoting endocytosis as in the beta(2) isoform. Major shifts in the IC for CGP12177 were also observed for T398 and T449. Even the removal of only 18 amino acid residues (T465) permitted a small but reproducible decrease in CGP12177 potency in response to agonist, between 1.2- and 1.8-fold in 6 separate experiments. However, some carboxyl-terminal structure is apparently required for endocytosis because complete removal of the carboxyl-terminal domain beyond the palmitoylated cysteine residue (T359) produced a receptor that was not endocytosed. Although the truncation mutants and the hamster beta(2)-adrenergic receptor were usually expressed at higher levels than the wild-type turkey beta(1)-adrenergic receptor, endocytosis behavior did not vary notably among L cell clones that expressed the same receptor at different levels. Endocytosis therefore does not reflect only the amount of receptor present or the relative stimulation of adenylate cyclase upon exposure to agonist.

Addition of the extreme carboxyl-terminal domain of the turkey beta(1)-adrenergic receptor to the body of the beta(2) receptor markedly diminished the effect of exposure to agonist. Exposure decreased the IC for CGP12177 by about 3-fold, much less than observed with the wild-type beta(2)-adrenergic receptor but still clearly altered. Unlike the other wild-type and mutant receptors, competition by CGP12177 for ICYP binding to the chimera was usually multiphasic after exposure of cells to agonist (see Fig. 2, for example), and at least one L cell clone displayed anomalously high affinity for CGP12177 with or without prior exposure to isoproterenol (not shown). Thus, addition of the carboxyl-terminal extension of the turkey beta(1)-adrenergic receptor to the beta(2) receptor significantly blocked its endocytosis, but other anomalies in its behavior made the effect less clear cut than was the case with the truncations.

An alternative assessment of agonist-promoted endocytosis comes from analysis of the distribution of receptors among cellular membrane fractions after their separation by sucrose density gradient centrifugation. As shown in Fig. 3, the endocytosis of beta(2)-adrenergic receptors is reflected by movement of receptors from a high-density peak, characteristic of plasma membrane fragments, to a low-density peak that is characteristic of endosomes (4, 38) . In addition to the change in sedimentation, receptors in the endosome fraction are relatively insensitive to the hydrophilic beta-adrenergic antagonist CGP12177, presumably because their ligand-binding sites face the lumen of the endosomes. Thus, beta(2)-adrenergic receptors from cells that were not exposed to agonist fractionated primarily at high density, and ICYP binding in this fraction was blocked 90% by 1 µM CGP12177. In contrast, 55% of the beta(2)-adrenergic receptors from cells that were exposed to isoproterenol were found in the endosome fraction. For these receptors in the low-density peak, CGP12177 blocked ICYP binding by only 18%. As shown previously (18, confirmed in Fig. 3), the turkey beta(1)-adrenergic receptor did not undergo agonist-promoted endocytosis according to this assay. The turkey beta(1)-adrenergic receptors remained in the high-density peak after treatment of cells with isoproterenol, and those receptors remained sensitive to CGP12177. According to this assay, T424 receptors behaved like mammalian beta(2)-adrenergic receptors: they moved to the endosome fraction and were protected from CGP12177. In contrast, the chimeric receptors behaved like turkey beta(1)-adrenergic receptors and remained in the plasma membrane fraction.


Figure 3: Effect of isoproterenol treatment on sedimentation of wild-type and mutant beta-adrenergic receptors. Untreated cells (squares) or cells exposed to isoproterenol for 20 min (circles) were lysed and lysates were fractionated on sucrose gradients as described under ``Experimental Procedures.'' The binding of [I]ICYP to receptors in gradient fractions was determined in the presence (open symbols) or absence (closed symbols) of 30-60 nM CGP12177. Data points are averages of triplicate assays performed in the presence and absence of 1 µM (-)-propranolol to determine specific receptor binding.



As summarized in Fig. 4for all mutant and wild-type receptors, the distal carboxyl-terminal domain of the turkey beta(1)-adrenergic receptor predominantly determined the migration of receptors from the plasma membrane fraction to the endosome fraction when cells were exposed to isoproterenol. Fig. 4shows the ratios of receptors found in endosomes and plasma membranes with and without exposure of the cells to isoproterenol. For every construct tested, fewer than 15% of the receptors were endosomal unless the cells were exposed to isoproterenol. After exposure to isoproterenol, both the hamster beta(2)-adrenergic receptor and three truncated turkey beta(1)-adrenergic receptors (T397, T424, and T449) were largely endocytosed. The wild-type beta(1)-adrenergic receptor and the chimera were not detectably endocytosed. Only a minority of the T359 and T465 receptors were found in endosomes, but a low level of endocytosis was noted reproducibly. Thus, the intact carboxyl terminus of the turkey beta(1)-adrenergic receptor can block endocytosis even of a beta(2)-adrenergic receptor, and the behavior of the truncation mutants indicates that 18 amino acid residues is the minimum amount necessary for this function. Note that the calculations used to generate the data shown in Fig. 4underestimate the sizes of both pools of receptors. However, calculating the ratios without reference to sensitivity to CGP12177 did not alter the overall pattern depicted in the figure.


Figure 4: Effect of isoproterenol treatment on the distribution of wild-type and mutant beta-adrenergic receptors between endosomal vesicles and plasma membrane. Control cells (hatched bars) or cells exposed to isoproterenol (INE) for 20 min (open bars) were lysed and analyzed by sucrose density gradient centrifugation as described in the legend to Fig. 3and under ``Experimental Procedures.'' Receptors in the endosomal vesicles were defined as [I]ICYP binding activity in the low-density fraction that was not blocked by 30-60 nM CGP12177. Receptors in the plasma membranes were defined as [I]ICYP binding activity in the high density fraction that was blocked by CGP12177. Data shown are means of 2, 3, 5, 4, 3, 3, 4, or 4 complete experiments (left to right). Error bars indicate S.E. values for isoproterenol-treated cells only. S.E. values for control cells varied from 0.01 to 0.07 (not shown).



Although the turkey beta(1)-adrenergic receptor is not endocytosed or down-regulated in response to agonist, it does undergo homologous desensitization in the form of uncoupling from G protein (16) . The data in Table 2indicate that truncating even the entire carboxyl-terminal region, in the T359 mutant, did not markedly diminish uncoupling. Although desensitization varied among experiments, its extent was generally 25-50% for both wild-type and mutant receptors. Likewise, attachment of the carboxyl-terminal region of the turkey receptor to the hamster beta(2)-adrenergic receptor did not markedly influence its desensitization.


DISCUSSION

Agonist-promoted internalization is a common property in the G protein-coupled receptor family, but the precise sequence motifs within these receptors that control internalization are not well defined. Diverse sequences in the cytoplasmic end of the seventh helix, the carboxyl-terminal domain, and the third cytoplasmic loop have all been implicated in initiating, inhibiting, or allowing endocytosis of receptors(2, 3) . The present study defines two regions in the carboxyl-terminal domain of the avian beta(1)-adrenergic receptors that are involved with its endocytosis. First, the endocytosis-incompetent phenotype of the T359 receptor confirms previous indications that the region just beyond the palmitoylated cysteine residue is required for endocytosis in response to agonist(6, 22, 24) . Second, the region between residues 450 and 465 blocks endocytosis in a receptor that is otherwise endocytosis-competent. The existence of a discrete endocytosis-blocking domain has been defined only in the avian beta(1)-adrenergic receptor(18, 28) , and this domain accounts for the inability of this receptor to be endocytosed(16, 17, 18) .

The mechanism whereby the carboxyl-terminal region prevents endocytosis remains unclear. When these same truncation mutants are expressed in Sf9 cells, we found that several other phenotypes coincided with restoration of endocytosis(28) . Although their intrinsic affinities for beta-adrenergic ligands are unaltered, the truncation mutants are more active stimulators of adenylyl cyclase in Sf9 cell membranes than are wild-type receptors. They stimulate somewhat in the absence of agonist, produce greater maximal activities in the presence of agonists, and are strikingly more sensitive to weak partial agonists. Their affinities for agonists are also more sensitive to guanine nucleotides. Last, the truncated receptors are expressed at higher levels and are more easily solubilized by mild detergents than are the wild-type receptors, which predominantly remain in the pellet after extraction with digitonin, dodecyl maltoside, or several other detergents(28) . These diverse effects led us to suggest that the carboxyl-terminal region may anchor the receptors either to cytoskeleton or to some other detergent-insensitive structure, such as caveolae. Such anchorage would both prevent their movement to coated pits, the site of endocytosis, and limit their access to G proteins. Several G proteins are found in caveolae, and the behavior of several G protein-coupled receptors is strongly influenced by drugs that interact with the cytoskeleton(35, 36, 37) .

The putative anchoring region at the carboxyl terminus of the turkey beta(1)-adrenergic receptor evidently constitutes an independent structural and functional domain within the protein. Its removal from the turkey receptor does not damage receptor function, and its addition to the hamster beta(2)-adrenergic receptor confers the phenotypes characteristic of the turkey receptor. According to analysis by sucrose density gradient centrifugation, the HTCR was not endocytosed at all in response to agonist ( Fig. 3and Fig. 4). Its behavior in the CGP12177 competition experiments also indicated marked blockade of endocytosis, although interpretation of the data was not straightforward (Fig. 2, Table 1). From the biphasic shape of the competition curve shown in Fig. 2and observed in other experiments, it appears that endocytosis of most of the receptors was totally blocked, but that a minority of HTCRs were protected from CGP12177. This might indicate that a few HTCRs are mis-sorted in the cell or that some undergo intracellular proteolysis to lose the anchorage domain.

The functional independence of the carboxyl-terminal anchorage domain is particularly striking because it is encoded by a 3` exon separate from the exon that encodes the rest of the receptor. The receptor RNA is alternatively spliced to yield either the long (full-length) receptor or a short receptor that is equivalent to T424(34) . Thus, a cell can use a single gene to express beta(1)-adrenergic receptors that either are or are not endocytosed in response to continued exposure to agonist.

The experiments reported here specify a small region of the turkey beta(1)-adrenergic receptor that inhibits agonist-promoted endocytosis. Removal of only 18 carboxyl-terminal amino acids permitted some endocytosis, as measured either by increased protection of the receptors from inhibition by the hydrophilic antagonist CGP12177 (Fig. 2, Table 1) or by movement of receptors to small, sealed vesicles (Fig. 4). Complete restoration of endocytosis was observed in the T449 mutant, in which 34 amino acid residues were removed, and little further effect was observed in T424 or T397. The region between 450 and 470 is therefore primarily responsible for anchoring the receptor in the plasma membrane. The small amount of endocytosis of the T465 mutant might reflect partial loss of the relevant structure, but it seems more likely that the important region lies between residues 449 and 465 and that the T465 phenotype reflects improper folding of that region. This conjecture is intuitively supported by the finding that residues 449-467 are conserved in the 3` open reading frames of the monkey and rat beta(1)-adrenergic receptor genes(34) . The sequence of this region is unremarkable and has only slight homology to other known proteins(34) . Regardless, the mapping of the anchorage domain in the present study is now fine enough to allow site-directed mutagenesis to determine its important structures.

In addition to their failure to endocytose, turkey beta(1)-adrenergic receptors are not down-regulated in response to agonist(16, 17, 18) . However, a spontaneous mutant that has lost the carboxyl-terminal domain is both efficiently endocytosed and down-regulated(18) . This finding suggests that the anchorage domain blocks down-regulation because it blocks endocytosis, a likely precursor to receptor degradation(2) . In many cells, however, down-regulation reflects both synthesis and degradation of receptors. Because the synthesis of recombinant receptors under the control of plasmid-born regulatory elements may not provide a valid image of physiological regulation, we have not studied down-regulation of the truncation mutants. On the other hand, the retention of agonist-promoted desensitization by all of the mutants (Table 2), which is characteristic of avian beta(1)-adrenergic receptors, argues that the carboxyl terminus of the beta(1)-adrenergic receptor is not required for receptor-G protein uncoupling. The carboxyl-terminal domain thus restricts long term removal of receptors by endocytosis and degradation while allowing desensitization mediated by uncoupling from G protein.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM 30355 (to E. M. R.) and GM36254 (to J. P. P.) and by R. A. Welch Foundation Grant I-0982 (to E. M. R.). 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.

§
Present address: Dept. of CNS Biology, Dept. 404, Bristol-Myers Squibb Co., 5 Research Pkwy., Wallingford, CT 06492.

Present address: Canji, Inc., San Diego, CA.

**
To whom correspondence and reprint requests should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041.

(^1)
The avian beta(1)-adrenergic receptor differs slightly from mammalian beta(1)-adrenergic receptors in its selectivity among some synthetic ligands(39) , but it is clearly of the beta(1) subtype according to its sequence, the organization of its gene(34) , and its overall pharmacologic specificity.

(^2)
The abbreviations used are: ICYP, (-)-iodocyanopindolol; HTCR, chimeric hamster beta(2)-adrenergic receptor to which the carboxyl-terminal region of the turkey beta(1)-adrenergic is appended.


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