©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Carboxyl-terminal Anchorage Domain of the Turkey -Adrenergic Receptor Is Encoded by an Alternatively Spliced Exon (*)

(Received for publication, November 28, 1994)

Jun Wang 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
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The originally described cDNA of the turkey beta(1)-adrenergic receptor encodes a receptor with a carboxyl-terminal, 59-amino acid extension that was not found in several mammalian beta(1)- adrenergic receptors. This extension blocks agonist-promoted endocytosis and down-regulation of the receptor. This carboxyl-terminal domain is encoded by an exon distinct from that which encodes the body of the receptor, and the originally described cDNA results from removal of an 849-nucleotide intron. Unspliced mRNA encodes a shorter open reading frame whose translated carboxyl terminus is identical with that of the mammalian beta(1)-adrenergic receptors. There is no evidence for other introns in the coding region. Splicing of the intron to produce the nonendocytosing receptor is highest in fetal blood cells, is appreciable in adult brain and heart, and is detectable in other tissues. Thus, different tissues use alternative splicing to express beta-adrenergic receptors that either do or do not endocytose and down-regulate in response to agonist.


INTRODUCTION

G protein-mediated signaling pathways undergo a complex process of desensitization in response to continued exposure to agonist. Desensitization is typically composed of multiple processes, some that are unique to the receptor for the desensitizing agonist (homologous desensitization) and others that desensitize downstream signaling components utilized by multiple receptors (heterologous desensitization). A major mechanism of homologous desensitization is receptor down-regulation, a multistep process in which receptors are first endocytosed and subsequently degraded (see (1) for review). The mechanism of agonist-promoted receptor endocytosis has been the focus of considerable research since its initial observation for the beta(2)-adrenergic receptor by Perkins and co-workers(1, 2, 3, 4, 5) . Endocytosis and subsequent loss of beta(2)-adrenergic receptors have become a prominent model system for the study of this process.

In contrast to the relatively coherent literature on the endocytosis and down-regulation of beta(2)-adrenergic receptors, desensitization of beta(1)-adrenergic signaling has been less extensively studied, and the results have been less clear and consistent (see Refs. 1, 6, and 7 for overview). Some investigators have described ``typical'' patterns of endocytosis and down-regulation for beta(1)-adrenergic receptors, while others have found that beta(1)-adrenergic receptors are endocytosed poorly if at all(6, 7, 8, 9, 10, 11, 12, 13, 14) . The avian beta(1)-adrenergic receptor, commonly studied in turkey or pigeon erythrocytes, has been paradigmatic of nonendocytosing beta(1)-adrenergic receptors since its initial description by Simpson and Pfeuffer(15) . Although this receptor differs slightly from mammalian beta(1)-adrenergic receptors in its pharmacologic selectivity, it is clearly of the beta(1)-adrenergic subtype. (^1)The avian beta(1)-adrenergic receptor is not appreciably endocytosed or down-regulated, either in its cell of origin (15, 16) or when it is expressed in mammalian cells that are capable of supporting endocytosis of other receptors(17, 18) .

We recently found that the inability of the turkey beta(1)-adrenergic receptor to undergo agonist-promoted endocytosis is determined by a 59-amino acid carboxyl-terminal extension that is apparently not shared with mammalian beta(1)-adrenergic receptors(17, 18) . Deletion of 30-100 amino acids from the carboxyl terminus of the avian receptor allows efficient endocytosis comparable to that observed with the beta(2)-adrenergic receptor. Furthermore, addition of the extension to the hamster beta(2)-adrenergic receptor blocks its endocytosis(18) . Thus, the carboxyl-terminal extension acts as an independent functional domain to limit endocytosis. In addition, the extension diminishes the signaling efficacy of the receptor in membranes, but not after purification and reconstitution in phospholipid vesicles, and it also inhibits the ability of the receptor to be solubilized using nondenaturing detergents(19) . These behaviors caused us to speculate that the extension may act by anchoring the receptor to cytoskeleton, thus both inhibiting its solubilization and, in cells, blocking its ability to move to coated pits where it could be endocytosed.

The genes for mammalian beta(1)- and beta(2)-adrenergic receptors have been described as being without introns(20, 21, 22, 23, 24, 25) . We describe here the existence of an intron in the turkey beta(1)-adrenergic receptor gene whose removal, by tissue-specific splicing, appends the endocytosis-inhibiting domain to the body of the receptor. The nonspliced mRNA encodes a shorter receptor that terminates at the same point as do the mammalian beta(1)-adrenergic receptors and which shares carboxyl-terminal identity with those receptors over its last 6 amino acid residues. Thus, birds can regulate the endocytosis and down-regulation of beta(1)-adrenergic receptors by the variable addition of an endocytosis-inhibiting domain to the receptors.


EXPERIMENTAL PROCEDURES

Genomic DNA was purified from turkey liver according to Sambrook et al.(26) , and specific sequences were amplified by PCR (^2)using Taq polymerase (Promega). Reactions routinely contained the reaction buffer provided by the manufacturer, 60 µg/ml template DNA, 2 µM primers, 1.5 mM MgCl(2), and 0.2 mM dNTPs. For routine PCR of genomic sequences, DNA was denatured at 94 °C for 5 min, and polymerase was added at 85 °C. DNA was then amplified for 30 cycles: 66 °C for 45 s, 68-72 °C for 60 s, and 92 °C for 60 s. Sequences of all PCR primers used in this study are given in Table 1. For sequencing, PCR products were cut with appropriate restriction nucleases, ligated into M13mp18 or M13mp19, and sequenced (27) according to the instructions provided with Sequenase DNA polymerase (U. S. Biochemicals).



Total RNA was purified by the acid phenol-guanidinium isothiocyanate method of Chomczynski and Sacchi(28) , and mRNA was enriched by chromatography on oligo(dT)-cellulose (New England Biolabs) according to the manufacturer's directions. Any contaminating DNA was destroyed by incubating the mRNA (40 µg/ml) with RNase-free DNase I (Promega; 28 units/ml) for 20 min at 37 °C in the assay buffer recommended by the manufacturer. The mixture was then extracted three times with phenol-chloroform, and RNA was precipitated with ethanol.

For cDNA synthesis, poly(A) RNA was mixed with 0.4 µg of oligo(dT)-primer per µg of RNA in Promega reverse transcriptase buffer supplemented with 0.25 mM dNTPs. The mixture was heated for 3 min at 75 °C, chilled at 0 °C for 2 min, and incubated for 60 min at 37 °C with 200 units of Moloney murine leukemia virus reverse transcriptase and 25 units of RNasin per µg of RNA. Parallel incubations without reverse transcriptase were routinely performed as a control for possible DNA contamination (see text).

Routine RT-PCR began with about 10 ng of cDNA in 50 µl of 10 mM Tris-Cl (pH 8.5), 2.5 mM MgCl(2), 50 mM KCl, 8% dimethyl sulfoxide, 1 µM concentration of each primer, and 0.3 mM dNTPs. DNA was denatured at 98 °C for 4 min, 2.5 units of Taq DNA polymerase was added at 72 °C, and PCR was run for 40 cycles: 60 °C for 1 min, 72 °C for 2-3 min, and 94 °C for 1 min. Ethidium-stained gels routinely contained 5 µl of the PCR reaction.

RNA denatured in 50% formamide was analyzed on 1% agarose gels that contained 2.2 M formaldehyde, as described by Sambrook et al.(26) , and transferred to Zeta-Probe membranes in 20 times SSC. Double-stranded DNA probes, prepared either from cloned cDNA or from PCR products as described in the text, were labeled by the random priming method (29, 30) according to instructions in the Stratagene kit. Blots were probed at 42 °C in 50% formamide, 5 times SSPE, 1% SDS, 2 times Denhardt's solution, 0.2% NaPP(i), and 0.1 mg/ml denatured herring sperm DNA. Blots were rinsed in 2 times SSC plus 0.1% SDS and washed twice at 58 °C for 20 min in 0.2 times SSC, 0.1% SDS, and 0.1% NaPP(i). Autoradiographs were exposed at -85 °C for 20-40 h.


RESULTS

The Turkey beta(1)-Adrenergic Receptor Gene Contains a Single Intron

PCR was used to amplify segments of the gene that encodes the turkey beta(1)-adrenergic receptor using primers that complement the sequence in codons 392-400 and 475-483 (Fig. 1A). This region encodes a cytoplasmic, carboxyl-terminal domain that inhibits receptor endocytosis and down-regulation and which limits receptor solubilization by detergents (17, 18, 19) . PCR yielded a predominant product of 1180 bp, much longer than the 330-bp product predicted by the cDNA sequence. The PCR product contained two PstI sites, also not found in the cDNA, such that digestion with PstI produced fragments of 520 bp, 440 bp, and 220 bp. These fragments were cloned into M13mp18 and M13mp19, and the DNA sequence was determined for inserts in duplicate phage isolates. Subsequently, a similar PCR product was prepared using primers that contained only BamHI sites. This 1180-bp product was again cloned, and its sequence around the internal PstI sites was determined. The balance of the original sequences was also confirmed (Fig. 1B).


Figure 1: A, PCR amplification of the beta(1)-adrenergic receptor gene from turkey genomic DNA. PCR was performed as described in the text using primers TS5 and TA7 (see Table 1). The product was analyzed by ethidium-stained agarose gel electrophoresis. Lane 1 shows the primary 1100-1200-bp product next to a commercial 100-bp ladder. When this product was digested with PstI on a larger scale and analyzed similarly, three bands (520 bp, 400 bp, and 220 bP) appeared (lane 2). The faint band just below 300 bp in lanes 1 and 2 may reflect nonspecific PCR priming. B, sequence of the intron in the turkey beta(1)-AR gene (GenBank accession number U14958), with surrounding genomic DNA sequence (lower case) shown for orientation. Numbering of the bases follows that used by Yarden et al.(31) , where the initiation codon begins with residue 70. The intron itself is bracketed, the two PstI sites are underlined, and the three potential polyadenylation signals are bold. Encoded amino acid sequences (in the two exons, and the valine and termination codons in the intron) are shown below the DNA sequence. X, nucleotides that were ambiguous or unreadable in multiple sequencing reactions using at least three M13 clones.



As shown in Fig. 1, the sequence of the genomic PCR products indicates that the turkey beta(1)-adrenergic receptor gene contains an 849-bp insert between codons 423 and 424 of the previously described cDNA sequence. The insert is flanked by consensus splice sites. Because the original cDNA sequence was obtained from three different cDNA clones isolated from two libraries, the 849-bp insertion in the gene evidently represents an intron that is removed during mRNA processing. The intron contains three canonical polyadenylation sites, although they are evidently not used to terminate the mRNA in those tissues that we have examined (see below).

No other introns were detected in the coding or 3` noncoding regions of the gene, either by PCR amplification of genomic DNA (not shown) or by RT-PCR of mRNA (Fig. 2A), although very short introns may have been missed by these approaches. We did not study the 5` noncoding region.


Figure 2: RT-PCR amplification of beta(1)-adrenergic receptor mRNAs from several turkey tissues. A, locations of the primers used for RT-PCR amplification of beta(1)-adrenergic receptor mRNA from turkey tissues and summary of successful reactions. ORFs are shown as solid areas in the linear sequence. RT-PCR reactions that yielded products of the expected sizes and with correct restriction sites are shown as heavy horizontal bars, and products that omit the intron sequence (i.e. that are amplified from spliced mRNA) are shown with dashed line inserts. B, dependence of RT-PCR reactions on reverse transcriptase (RT). As a control for DNA contamination of mRNA, duplicate samples of fetal blood mRNA were amplified by RT-PCR under identical conditions but in which one reaction lacked RT. A 100-bp ladder is shown at the left, with the 800-bp marker noted by an arrow. C, RT-PCR of mRNA from several turkey tissues designed to show amplification of both spliced and unspliced mRNA (TS6-TA6; lane 1); unspliced mRNA between exon I and the intron (TS6-TA5; lane 2); and unspliced mRNA between the intron and exon II (TS8-TA6; lane 3).



The intron in the turkey gene occurs just after the codon 423 (lysine), where protein sequence is strongly conserved among all beta(1)-adrenergic receptors(22, 23, 24) . Mammalian beta(1)-adrenergic receptors terminate with a valine immediately after this lysine, but the turkey beta(1)-adrenergic receptor cDNA (31) encodes a methionine at position 424 followed by a 59-amino acid extension. In the turkey genomic DNA sequence (Fig. 1B), codon 423 (lysine) is followed immediately by a valine codon and termination codon. Thus, an unspliced mRNA in the turkey would encode a shortened receptor that is identical in its carboxyl-terminal sequence with the mammalian receptors. Such a short receptor is efficiently endocytosed in response to agonist(17, 18) . Removal of the intron by RNA splicing produces the originally described 483-codon turkey receptor with the carboxyl-terminal anchorage domain that is unique among the beta(1)-adrenergic receptors. We therefore decided to study the expression of both potential products of the receptor gene.

The Intron in the Turkey beta(1)-Adrenergic Receptor mRNA Is Differentially Removed by Alternative Splicing

The presence of spliced and unspliced receptor mRNA was initially detected by RT-PCR of mRNA prepared from tissues that, based on prior studies with mammals, are expected to express beta(1)-adrenergic receptors. Several primer pairs were used to detect the presence or absence of the intron in the mRNA. Initial studies were performed on poly(A) RNA purified from fetal blood, the source of the RNA used in the initial cloning of the cDNA(31) , and all reverse transcriptase reactions were primed with oligo(dT). As shown in Fig. 2, A and B, RT-PCR of receptor mRNA was successful throughout the coding and 3` noncoding regions. RT-PCR of mRNA from multiple tissues indicated the presence both of unspliced mRNA and of the spliced RNA that corresponds to the cDNA in which the entire intron was removed.

Spliced mRNA was detected using primer pairs on either side of the intron (Fig. 2, A and B). The products, which were obtained using multiple primer pairs, were all consistent with the existence of a single spliced mRNA according to length and to the presence of diagnostic restriction sites. Minor products, including slightly shorter fragments, were observed frequently, and are assumed to represent mispriming in early PCR cycles.

Unspliced mRNA was detected most readily when one or both primers contained intron sequence. Fig. 2B shows products obtained that are totally within the intron, that extend from exon I into the intron or from the intron into exon II. Multiple primer pairs were tested with similar results (Fig. 2A). Only one primer pair, TS6/TA6, reproducibly yielded a RT-PCR product that completely spanned the intron, beginning in exon I and ending in exon II (Fig. 2B). This primer pair amplified both the spiced and unspliced mRNAs, and the shorter spliced form was always favored even when the elongation time of the PCR cycle was extended to 4 min. Such preferential amplification of shorter templates by PCR is commonly observed. Note that for all of the RT-PCR reactions, the amount of product could be altered markedly by changing the conditions of the PCR reaction, particularly the elongation time, and the amount of product in a given reaction cannot therefore be taken to indicate the relative amount of splicing.

Although the intron in the beta(1)-adrenergic receptor gene contains three consensus polyadenylation sequences, a major fraction of the unspliced mRNA appears to terminate at residue 2655, the same point as does the spliced product. Thus, RT-PCR amplification of unspliced mRNA sequences 3` to the first three potential polyadenylation sites (using TA6, TA7, or TA9 as the antisense primer) was as efficient as that which terminates at more 5` sites measured only by primers TA3 or TA4). Termination of the spliced mRNA at residue 2655 was confirmed by Northern analysis, below.

RT-PCR products were not the result of contamination of the purified RNA either with cDNA or with genomic DNA. First, all RNA samples were treated with DNase before the reverse transcriptase reaction (see ``Experimental Procedures''). In addition, the RT-PCR reaction was blocked by destruction of the mRNA template with DNase-free RNase (not shown). Last, amplification of all sequences was dependent on reverse transcriptase to synthesize the first DNA strand before PCR (Fig. 2B). Control data of the sort shown in Fig. 2B were routinely obtained for all primer pairs, for multiple preparations of mRNA from several tissues, and for multiple subsequent syntheses of cDNA.

Spliced and Unspliced beta(1)-Adrenergic Receptor mRNA Is Found in Multiple Tissues

RT-PCR was used to screen multiple tissues expected to have beta(1)-adrenergic receptors for the two forms of the mRNA. As shown in Fig. 2, all tissues tested displayed both forms of the mRNA. Differences in the amounts of the long product (unspliced template) and the short product (spliced template) were not striking. Furthermore, the relative yield of each product was quite sensitive to the conditions used for amplification. Consequently, semiquantitative comparison was undertaken by Northern analysis.

Northern blots of mRNA prepared from turkey tissues showed two major mRNA species that hybridized with beta(1)-adrenergic receptor probes. One had a molecular size of about 1800 bp, the other about 2500 bp. These sizes are predicted by the use of the distal polyadenylation signal at nucleotide 2640, with or without removal of the 849-bp intron. Consistent with this interpretation, the 2500-bp mRNA species hybridized to probes based on the first exon, the intron, or the second exon. Its size also corresponded to the predicted value within the resolution of agarose gel electrophoresis. The 1800-bp mRNA was of the size predicted by the original cDNA(31) . It hybridized with exon-specific probes but not with an intron probe (Fig. 3A).


Figure 3: Northern blots of turkey beta(1)-adrenergic receptor mRNA. mRNA was prepared, electrophoresed, and transferred to nylon membranes as described in the text. A, triplicate blots of mRNA from heart (4 µg) and fetal blood (2 µg) were hybridized to probes specific for the first exon (283-1020), the intron (1766-2162), or the second alternatively spliced exon (2020-2367; in the ORF that encodes the carboxyl terminus of the long form of the receptor). Probes were prepared by random-primed labeling of gel-purified restriction fragments of cDNA or PCR products. Molecular sizes at the left show the locations of Promega RNA size markers. B, tissue distribution of the large (unspliced) and small (spliced) turkey beta(1)-adrenergic receptor mRNAs. mRNA samples from the tissues shown (3 µg/lane for fetal blood; 8 µg/lane for other tissues) were analyzed by Northern blotting using a probe for the 5` region of the receptor ORF. Autoradiographs were exposed for different times to facilitate comparison of the long and short mRNAs, and absolute intensities should not be taken to represent the abundance of total receptor mRNA.



Northern analysis of mRNA from multiple tissues displayed great differences in the relative splicing of the receptor mRNA. The highest ratio of spliced to unspliced mRNA was found in fetal blood, the source of the initial cDNA clone (Fig. 3B). The spliced product was usually equal to or more abundant than the unspliced. At the other extreme, gizzard and skeletal muscle produced primarily the unspliced product (3% spliced, near background). Heart, bone marrow, brain, and lung were intermediate between these extremes, producing 10-30% spliced mRNA (Fig. 3B). It is unlikely that the spliced RNA in these tissues reflects contamination by blood because mature avian erythrocytes, although nucleated, are transcriptionally inactive and contain little mRNA. The blood of mature turkeys contains few immature erythroid cells (32) (confirmed by examination of May-Grunwald-Giemsa-stained smears(32) , not shown).

For most tissues, the ratios of spliced to unspliced mRNAs were reproducible among multiple preparations of mRNA and multiple syntheses of cDNA. Thus, alternative splicing of the beta(1)-adrenergic mRNA may determine the ability of the receptor to undergo agonist-promoted endocytosis in different tissues. In multiple samples of bone marrow, however, the ratio of spliced to unspliced mRNA was highly variable, ranging from almost undetectable to about equal amounts of the two forms. Bone marrow was obtained from turkeys freshly killed in a commercial slaughterhouse, and we do not know if this variability reflects varied age, health, sex, or nutritional history. Variable splicing does suggest marked physiological control of the two forms of the receptor in blood cells.

Homology between the 3` Regions of the Mammalian and Turkey beta(1)-Adrenergic Receptor Genes Does Not Dictate Common Splicing of the Rat mRNA

Previously described cDNAs for mammalian beta(1)-adrenergic receptors are structurally similar to the unspliced form of the turkey beta-adrenergic receptor(22, 23, 25) , and Northern blots of total RNA from various rat tissues all displayed a predominant band of about 2500 bp(22) . However, 3` regions of the beta(1)-adrenergic receptor gene in both rat and rhesus monkey are strikingly similar to the 3` portion of the intron and the second exon of the turkey beta(1)-adrenergic receptor gene (Fig. 4A). Immediately after the first termination codon, avian and mammalian genes display some homology for about 100 bp, but are then dissimilar for about 700 bp. At that point, there begins extensive homology between the turkey, rat, and monkey genes that extends for at least 600 bp, which is the extent of sequence that is available for the turkey. The mammalian genes also contain an ORF that encodes protein sequence homologous to the carboxyl-terminal domain of the avian receptor (Fig. 6). Particularly in the monkey gene, this ORF is positioned to allow in-frame splicing to encode a carboxyl-terminal extension, albeit with a relatively poor consensus 3` splice site.


Figure 4: RT-PCR analysis to detect possible splicing of the rat beta(1)-adrenergic receptor mRNA. A, alignment of the turkey and rat beta(1)-adrenergic receptor genes. Numbering of the rat gene is according to Shimomura and Terada (24) as extended by Searles et al.(23) . Homology is extensive over the first ORF (double-hatched bar) except for the amino terminus and third cytoplasmic loop, which account for the difference in lengths. The two genes are also quite similar from just after residue 2000 in the turkey gene through the end of the available turkey sequence (single-hatched bar). The locations of rat PCR primers (labeled arrows; see Table 1) are shown below the map. Below the primers are the products that were successfully amplified by RT-PCR of rat mRNA. All products were of the predicted length based on the assumption of no splicing. B, representative RT-PCR products from mRNA prepared from different rat tissues. Lane 1, RS3-RA2; lane 2, RS3-RA3; lane 3, RS3-RA6; lane 4, RS3-RA7; lane 5, RS4-RA5. Note that RA6 primes just before the polyadenylation site and that RA7 primes just after this site, and the absence of a product with RA7 indicates the termination site of the mRNA. Molecular sizes are shown at left.




Figure 6: Comparison of the amino acid sequence encoded by ORFs in the 3` regions of the beta(1)-adrenergic receptor genes in turkey (Tur)(31) , rat(24) , and rhesus monkey (Mon)(23) . Overlap extends from Gly through His in the turkey receptor. In the 25-residue region of overlap, sequence identity is 48% for turkey/monkey, 36% for turkey/rat, and 32% for rat/monkey. The monkey ORF begins with a clear, in-frame consensus splice acceptor site (CAG/A), but a site for in-frame splicing is not obvious in the published rat gene sequence. Truncation after Phe, but not after Thr, confers the endocytosis-competent phenotype(18, 19) .



We have attempted to determine whether the rat beta(1)-adrenergic receptor mRNA is also spliced to link the 3` ORF to the carboxyl terminus of the receptor. Initial Northern blots of mRNA from a few rat tissues only displayed the single unspliced mRNA (Fig. 5), as described previously by Frielle et al.(22) . We therefore used RT-PCR to test for spliced mRNA in several different tissues using several primer pairs that spanned the putative intron. However, we found no evidence of splicing (Fig. 4B and Fig. 5). All RT-PCR reactions yielded products consistent with unspliced mRNA. Thus, the rat beta(1)-adrenergic receptor mRNA is not spliced in the tissues studied here.


Figure 5: Northern analysis of beta(1)-adrenergic receptor mRNA from rat tissues. mRNA was prepared as described under ``Experimental Procedures,'' and 6-µg samples were electrophoresed and transferred to nylon membranes. Blots were hybridized with labeled probe specific for the first ORF (1826-2633).




DISCUSSION

The first genes for G protein-coupled receptors to be cloned lacked introns in their coding regions, and introns are generally few in this family. However, alternative splicing to alter receptor structure has been described for several of the G protein-coupled receptors(33, 34, 35, 36, 37, 38, 39) , and such splicing can alter the receptor's efficacy and G protein selectivity(40, 41, 42, 43, 44) .

We now find that the turkey beta(1)-adrenergic receptor gene contains an alternatively spliced intron. Removal of the intron from the mRNA appends a 59-amino acid, carboxyl-terminal extension to the receptor and thereby blocks the receptor's ability to undergo agonist-promoted endocytosis. Failure to remove the intron produces an mRNA that encodes a shorter receptor that can be endocytosed. Previously described mammalian beta(1)- and beta(2)-adrenergic receptor cDNAs lack this extension, and these receptors undergo endocytosis (see (1) and (18) and the references cited therein). Addition of the extension to the hamster beta(2)-adrenergic receptor blocks its endocytosis(18) .

The spliced and unspliced receptor mRNAs use the same polyadenylation signal ( Fig. 3and Fig. 4). In the unspliced mRNA, continued translation into the ``intron'' of the alternatively spliced form terminates the receptor after Val, which will allow its endocytosis in response to agonist. This carboxyl terminus, Ser-Glu-Ser-Lys-Val, is conserved among all of the mammalian beta(1)-adrenergic receptors whose cDNAs have been cloned. Northern analysis and RT-PCR detected only these two splice products of the turkey beta(1)-adrenergic receptor gene, although minor variants might be undetected or exist in tissues not tested here.

The ratio of spliced to unspliced beta(1)-adrenergic receptor mRNA varied markedly among the tissues tested here. In fetal blood, from which the spliced cDNA was first cloned, there was consistently more spliced than unspliced mRNA. Among the other tissues tested, heart, lung, and brain yielded the highest ratio of spliced to unspliced mRNA, and in situ RT-PCR of brain sections might be of significant neurological interest. Still other tissues displayed little splicing. In contrast to the fetal blood, measurement of spliced and unspliced receptor mRNA from bone marrow has yielded variable results. A few preparations have shown substantial amounts of the spliced mRNA, but others contained primarily the unspliced form (Fig. 3). The source of the variability is unknown.

The splicing of beta(1)-adrenergic receptor mRNA allows cells to express receptors that either do or do not undergo endocytosis and degradation in response to continued exposure to agonists. It is tempting to speculate that, in erythrocytes, which are long-lived but are translationally inactive, desensitization of beta-adrenergic signaling must involve reversible events instead of receptor endocytosis, which typically precedes receptor degradation(1) . Expression of the nonendocytosing form of the receptor in heart or brain is less easy to rationalize. It may reflect the need of some cells in these tissues to regulate signaling either only transiently or only through heterologous mechanisms. It has also been proposed that endocytosis of beta(1)-adrenergic receptors followed by recycling to the cell surface allows receptor dephosphorylation and consequent resensitization(46) . The nonendocytosing form of the receptor may thus resensitize more slowly. This question can probably best be answered in the heart, where there is a significant amount of both forms of the receptor and where beta-adrenergic physiology is best understood.

The introduction of discrete structural and functional domains is thought to be a major use of exons both in evolution and in development. As described elsewhere, the carboxyl-terminal extension encoded by the second exon of the spliced receptor mRNA acts as a distinct structural and functional element both in the turkey beta(1)-adrenergic receptor and when it is appended to the mammalian beta(2)-adrenergic receptor. It blocks endocytosis, inhibits solubilization by nondenaturing detergents, and limits the ability of the receptor to regulate the G(s)-adenylyl cyclase system in membranes. We have speculated that binding to cytoskeleton or some other fixed cellular structure can account for all three effects (19) .

Alternative splicing of the avian beta(1)-adrenergic mRNA explains the previous confusion over the carboxyl-terminal structure of the native receptor in turkey erythrocytes. The first cloned cDNA for this receptor was a product of the spliced mRNA and thus encodes the long, nonendocytosing receptor. However, immunoblot analysis with peptide-directed antibodies indicated that the beta-adrenergic receptor purified from turkey erythrocytes terminates near residue 424, the junction of the two exons(47) . Such a short receptor should be endocytosed, which is not consistent with its behavior in cells(15, 17) . These observations are explained by the fact that turkey erythrocytes display both long and short receptors. The short form, apparently in the minority, is more readily solubilized with detergent and is thus enriched during purification.

Is the mRNA for the Mammalian beta(1)-Adrenergic Spliced?

The 3` regions of the rat and rhesus monkey beta(1)-adrenergic receptor genes are strikingly similar in sequence from the latter part of the intron through the second exon and the 3` noncoding region of the turkey gene (Fig. 4A). Homology includes the conservation of donor and acceptor splice sites such that, at least in the monkey, in-frame splicing could append a carboxyl-terminal extension that is homologous to the receptor-anchoring domain of the turkey (Fig. 6). Specifically, the homologous sequence represents the region near the carboxyl terminus that is important for the control of endocytosis. Truncation before this sequence (after Phe) permits endocytosis and detergent solubilization, but truncation after Thr does not(18, 19) .

Alternative splicing of mammalian beta(1)-adrenergic receptor mRNA is attractive because it would clarify extensive disagreement about the endocytosis and down-regulation of this receptor (see introduction to the text). In some cultured cells and tissues, beta(1)-adrenergic receptors undergo efficient endocytosis and down-regulation, but other apparently careful studies have reported that beta(1)-adrenergic receptors are endocytosed slowly, partially, or not at all. Alternative splicing of the mRNA could account for such variability in behavior.

In this regard, we looked unsuccessfully for splicing of the rat beta(1)-adrenergic receptor mRNA. Given the sensitivity of RT-PCR, spliced mRNA should have been detected if it were present, and we conclude that the receptor mRNA is not spliced in the tissues and cells that we studied. It is of course possible that the mRNA is spliced in other species, and the rat genomic sequence has a poor 3` splice acceptor site. Species-specific slicing has been proposed for the beta(3)-adrenergic receptor(25) . Alternatively, other structural elements may modulate endocytosis in mammalian beta(1)-adrenergic receptors either independently or in concert with the putative carboxyl-terminal extension. For example, Suzuki et al.(7) transfected CHW cells with beta(1)-adrenergic receptor cDNA that contained only the major 5` ORF and found that the expressed receptors did not endocytose efficiently. It thus remains to be seen whether alternative splicing is a general mechanism for modulating the endocytosis and down-regulation of beta(1)-adrenergic receptors in mammals as it is in birds.


FOOTNOTES

*
This work was supported by a postdoctoral fellowship from Cadus Pharmaceuticals, Inc., National Institutes of Health Grant R37GM30355, and Robert A. Welch Foundation Grant I-0982. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14958[GenBank].

§
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)
Turkeys express at least three beta-adrenergic receptor subtypes: the beta(1) subtype discussed here, a beta(2) subtype(45) , and a third subtype denoted ``4C'' (which is probably not analogous to the mammalian beta(3); (45) ). The receptor described here is beta(1) based on the predominance of its pharmacologic specificity, although it differs somewhat from mammalian beta(1)-adrenergic receptors in its affinities for some synthetic ligands(48) . Its membrane spans and short interspan loops share 81% amino acid identity with mammalian beta(1)-adrenergic receptors but only 64% identity with the mammalian beta(2) isoform. (The mammalian beta(1) isoform is similarly 64% identical with the mammalian beta(2) in these regions.) Last, the overall structure of the turkey beta(1)-adrenergic receptor gene is strikingly similar to that of the rat and monkey beta(1)-adrenergic receptor genes (this work).

(^2)
The abbreviations used are: PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR amplification of cDNA synthesized from poly(A) RNA samples; ORF, open reading frame; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank David Russell, Mark Lehrman, and Tom Wilkie for advice and discussion throughout this study, Rob Nicholas (Univ. of North Carolina) for sending us his manuscript before its publication, and Belinda Sloan-Sanchez for technical assistance. Several pilot experiments on the rat mRNA were performed by Roanna Padre. We are grateful to Carolyn Overton and the staff of Plantation Foods, Waco, TX, for assistance in obtaining tissue samples.


REFERENCES

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