(Received for publication, November 28, 1994)
From the
The originally described cDNA of the turkey
-adrenergic receptor encodes a receptor with a
carboxyl-terminal, 59-amino acid extension that was not found in
several mammalian
- 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
-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
-adrenergic receptors that either do or do not endocytose and
down-regulate in response to agonist.
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 -adrenergic receptor by Perkins and
co-workers(1, 2, 3, 4, 5) .
Endocytosis and subsequent loss of
-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 -adrenergic
receptors, desensitization of
-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
-adrenergic receptors, while
others have found that
-adrenergic receptors are
endocytosed poorly if at
all(6, 7, 8, 9, 10, 11, 12, 13, 14) .
The avian
-adrenergic receptor, commonly studied in
turkey or pigeon erythrocytes, has been paradigmatic of nonendocytosing
-adrenergic receptors since its initial description by
Simpson and Pfeuffer(15) . Although this receptor differs
slightly from mammalian
-adrenergic receptors in its
pharmacologic selectivity, it is clearly of the
-adrenergic subtype. (
)The avian
-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 -adrenergic receptor to
undergo agonist-promoted endocytosis is determined by a 59-amino acid
carboxyl-terminal extension that is apparently not shared with
mammalian
-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
-adrenergic receptor. Furthermore, addition of the
extension to the hamster
-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
- and
-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
-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
-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
-adrenergic receptors by the
variable addition of an endocytosis-inhibiting domain to the receptors.
Genomic DNA was purified from turkey liver according to
Sambrook et al.(26) , and specific sequences were
amplified by PCR ()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
, 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, 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 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
SSPE,
1% SDS, 2
Denhardt's solution, 0.2% NaPP
, and
0.1 mg/ml denatured herring sperm DNA. Blots were rinsed in 2
SSC plus 0.1% SDS and washed twice at 58 °C for 20 min in 0.2
SSC, 0.1% SDS, and 0.1% NaPP
. Autoradiographs were
exposed at -85 °C for 20-40 h.
Figure 1:
A,
PCR amplification of the -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
-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
-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
-adrenergic receptor mRNAs from several turkey
tissues. A, locations of the primers used for RT-PCR
amplification of
-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 -adrenergic
receptors(22, 23, 24) . Mammalian
-adrenergic receptors terminate with a valine
immediately after this lysine, but the turkey
-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
-adrenergic receptors. We therefore decided
to study the expression of both potential products of the receptor
gene.
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 -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.
Northern blots of mRNA prepared from turkey
tissues showed two major mRNA species that hybridized with
-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
-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
-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
-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.
Figure 4:
RT-PCR analysis to detect possible
splicing of the rat -adrenergic receptor mRNA. A, alignment of the turkey and rat
-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 -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 -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
-adrenergic receptor mRNA is not spliced in the
tissues studied here.
Figure 5:
Northern analysis of
-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).
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 -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
- and
-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
-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
-adrenergic
receptors whose cDNAs have been cloned. Northern analysis and RT-PCR
detected only these two splice products of the turkey
-adrenergic receptor gene, although minor variants
might be undetected or exist in tissues not tested here.
The ratio
of spliced to unspliced -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
-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
-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
-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
-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
-adrenergic receptor and when it is appended to the
mammalian
-adrenergic receptor. It blocks endocytosis,
inhibits solubilization by nondenaturing detergents, and limits the
ability of the receptor to regulate the G
-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
-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
-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.
Alternative splicing of mammalian
-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,
-adrenergic receptors
undergo efficient endocytosis and down-regulation, but other apparently
careful studies have reported that
-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
-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
-adrenergic receptor(25) . Alternatively,
other structural elements may modulate endocytosis in mammalian
-adrenergic receptors either independently or in
concert with the putative carboxyl-terminal extension. For example,
Suzuki et al.(7) transfected CHW cells with
-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
-adrenergic receptors in mammals as it is in birds.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14958[GenBank].