(Received for publication, August 8, 1995; and in revised form, December 28, 1995)
From the
Homologous desensitization of G protein-coupled receptors
involves agonist-dependent phosphorylation of receptors by G
protein-coupled receptor kinases (GRKs). To identify GRK(s) that play a
role in homologous desensitization of the thyrotropin (TSH) receptor,
thyroid cDNA was amplified by polymerase chain reaction using
degenerate oligonucleotide primers from highly conserved regions in GRK
family. GRK5 is found in the predominant isoform expressed in the
thyroid. Rat GRK5 cDNA was then isolated, which encodes a 590-amino
acid protein with 95% homology to human and bovine homologs. Northern
blot identified GRK5 mRNA of 3, 8, and 10 kilobases with highest
expression levels in lung > heart, kidney, colon > thyroid. In
functional studies using a normal rat thyroid FRTL5 cells,
overexpression of GRK5 by transfecting the plasmid capable of
expressing the sense GRK5 RNA suppressed basal cAMP levels and
augmented the extent of TSH receptor desensitization, whereas
suppression of endogenous GRK5 expression by transfecting the antisense
GRK5 construct increased basal cAMP levels and attenuated the extent of
receptor desensitization. Although exogenously overexpressed GRK6 also
enhanced TSH receptor desensitization, we conclude that GRK5, the
predominant GRK isoform in the thyroid, appears to be mainly involved
in homologous desensitization of the TSH receptor.
Guanine nucleotide-binding regulatory (G) ()protein-coupled receptors transduce a wide variety of
extracellular signals (hormones, neurotransmitters, odorants, lights,
chemoattractants, etc.) into intracellular signaling events, by
activating or inhibiting specific effector enzymes (adenylyl cyclase,
phospholipase C, phospholipase A
, ion channels,
etc.)(1) . A rapid loss of responsiveness also occurs following
receptor activation by agonist ligand binding in this receptor
superfamily. This process is called ``homologous
desensitization.'' The mechanisms of homologous desensitization
have been extensively studied at the molecular level in rhodopsin and
adrenergic receptors (2, 3, 4) . Newly
discovered G protein-coupled receptor kinases (GRKs) and arrestins are
involved in this process. GRKs specifically recognize and phosphorylate
the agonist-bound form of receptors in the active conformation.
Subsequently, arrestin proteins bind exclusively to the phosphorylated
and activated receptor, resulting in uncoupling of the receptor and G
protein.
Six different types of GRKs have so far been cloned and
characterized. cDNA for bovine -adrenergic receptor kinase
(
-ARK, now also called GRK2) was first isolated(5) ,
followed by the cloning of cDNAs for bovine
-ARK2
(GRK3)(6) , bovine rhodopsin kinase (GRK1)(7) , human
IT11 (GRK4)(8) , human GRK5(9) , and human
GRK6(10, 11) . Drosophila kinases GPRK-1 and
-2 have also been identified(12) . These GRKs can be divided
into 3 different subgroups according to their homology, cellular
localization, and distinct regulatory
mechanisms(9, 10) . The first subgroup includes GRK4
to 6 and GPRK-1, the second subgroup GRK1, GRK2, and GPRK-2, and the
third subgroup GRK1. GRKs also seem to have different patterns of
expression in tissues. GRK1 and GRK4, for instance, are primarily
expressed in retinal cells and the testis, respectively, suggesting
very specific functions. GRK1 (rhodopsin kinase) phosphorylates
photolyzed rhodopsin (7) . Although little is at present known
about substrate specificity, the testis-specific expression of GRK4
implies that it might be a kinase for gonad-specific G protein-coupled
receptors such as the lutropin and follitropin receptors(3) .
Whereas, other GRKs are expressed in many tissues, suggesting their
broad roles in regulating many receptors.
The receptor for
thyrotropin (thyroid stimulating hormone, TSH), that regulates thyroid
growth and differentiated functions, is also a member of G
protein-coupled receptors (13) and well known to undergo
homologous, but not heterologous,
desensitization(14, 15, 16) . Although we
have recently reported that only the hormone-occupied form of the TSH
receptor, like the -adrenergic receptor, is likely to be involved
in homologous desensitization(17) , the molecular mechanisms of
TSH receptor desensitization remain largely unclear.
The aim of the present studies was, therefore, to characterize the GRK(s) that may play a role in TSH receptor homologous desensitization in the thyroid. Using reverse transcription and the polymerase chain reaction (RT-PCR), human and rat thyroid mRNA was studied to determine which types of GRK are present in these cells and whether these include any novel thyroid-specific GRK. We report herein that GRK5 appears to be the predominant GRK isoform in the thyroid cells. Furthermore, we have cloned and determined the nucleotide sequence of rat GRK5 cDNA of thyroid origin and provide evidence to support the functional role of GRK5 in homologous desensitization of the TSH receptor.
Figure 1: Deduced amino acid sequence of rat GRK5. Deduced amino acid sequence of the full-length rat GRK5 is shown. Predicted amino acid sequence is numbered on the right.
The human GRK6 cDNA in pBS which was kindly provided by Dr. Bonovic (10) was ligated into the expression vector pCR3. The transfection and selection with G418 were performed as described above.
The cells were cultured to semi-confluence in 10-cm dishes in 3H-medium. After washing three times with phosphate-buffered saline, pH 7.4, the cells were incubated with TSH-free medium for 5 days, and were then passed into 24-well culture plates. After 2 days, the cells were stimulated for the indicated periods at 37° C in the medium supplemented with 10 units/liter TSH and 0.5 mM 3-isobutyl-1-methylxanthine. In other experiments, the cells, cultured in the absence of TSH for 7 days, were preincubated for 4 h at 37° C with or without TSH, washed three times with phosphate-buffered saline, and incubated for two 30-min periods in medium without TSH to ensure the removal of TSH. The cells were then stimulated for 30 min at 37° C with the medium containing 10 units/liter TSH and 0.5 mM 3-isobutyl-1-methylxanthine. Intracellular cAMP concentrations were measured as previously reported (25) by radioimmunoassay (Yamasa cAMP kits, Yamasa Shoyu, Japan). Each experiment involved duplicate or triplicate wells and was repeated at least twice.
Using degenerate oligonucleotide primers corresponding to highly conserved sequences in the catalytic domain of members of the GRK family, the DNA fragments of the expected size (approximately 300 bp) were successfully amplified by PCR from human and rat thyroid cDNA. The PCR products were then subcloned into pBS and their nucleotide sequences were determined. As summarized in Table 1, of 30 cDNA clones obtained from a human thyroid, 27 were identical to human GRK5, 2 were GRK4, and -1 was GRK6. In the rat thyroid, of 20 cDNA clones sequenced, 19 and 1 showed approximately 90% homology to human GRK5 and -4, respectively, suggesting these may be rat GRK5 and 4. Rat GRK4 and -5 cDNAs have not previously been cloned. Neither GRK1, -2, nor -3 were observed in human and rat thyroids. No novel GRK mRNA species was identified.
Since these data indicate that the
predominant GRK isoform expressed in the thyroid is likely to be GRK5
and because a well characterized, normal rat thyroid epithelial cell
line, FRTL5 cells, is a very useful model for further functional
studies, we attempted to clone rat GRK5 cDNA. 4 10
recombinant phages of a rat FRTL5 cDNA library were screened with
the PCR fragment of the rat GRK5-like cDNA mentioned above. We isolated
four independent clones at high stringency condition. Partial
nucleotide sequencing of these four clones revealed that the 5`-ends of
all four clones were very similar, varying by no more than 20 bp (data
not shown). The nucleotide sequence of one cDNA clone with the longest
5`-end was determined in full (data not shown). The 2.3-kilobase
sequence consists of a single open reading frame of 1770 bp flanked by
183 bp of 5`-untranslated sequence and 365 bp of 3`-untranslated
sequence with a short stretch of poly(A) tract. The assignment of the
initiation codon ATG is based on the high degree of similarity of the N
termini of GRKs. Rat, as well as human and
bovine(9, 26) , GRK5 do not have a Kozak consensus
sequence for translation initiation(27) . The open reading
frame of GRK5 cDNA encodes a protein of 590 amino acids with a
predicted molecular mass of 68 kilodaltons. Deduced amino acid sequence
of rat GRK5 was shown in Fig. 1.
Comparison of this
nucleotide sequence and the deduced amino acid sequence with those of
the previously identified members of mammalian GRK family indicates
that this sequence is a rat homolog of human GRK5: 88.8% nucleotide
homology and 95.1% amino acid homology. While this work was in
progress, bovine GRK5 cDNA was isolated (26) and has a very
high homology with rat GRK5 (94.0% amino acid identity). As mentioned
in the Introduction, GRKs can be grouped into three distinct
subfamilies, GRK1, GRK2/3, and GRK4/5/6. The similarity between
GRK4/5/6 is 80%, that between GRK4/5/6 and GRK1/2
60%, and
that between GRK4/5/6 and GRK1 is intermediate (
70%). GRK5 has
some similarities to and differences with other GRKs. For instance, the
catalytic domain in the central region of the GRK5 contains a DLG
sequence, a feature characteristic of the GRKs. On the other hand, the
C-terminal domain does not possess a CAAX motif for
isoprenylation or G protein
/
-binding site(4) .
mRNA distribution in several tissues was then investigated by
Northern blot analysis (Fig. 2). The highest expression of GRK5
mRNA was observed in lung > colon, kidney, and heart > thyroid.
Absence of GRK5 message in liver is consistent with the previous
reports for human and bovine GRK5, but renal expression of GRK5 may be
unique in rat(9, 26) . Three different sizes of
messages, 3, 8, and 10 kilobases in length, were identified in all
cases, probably by use of alternative polyadenylation signals.
Alternatively, the 8- and 10-kilobase messages may be precursors of
mature mRNA. Rat thyroid RNA (20 µg of total RNA) demonstrated
little detectable bands for GRK4 and GRK6 even by longer exposure (data
not shown).
Figure 2:
Distribution of GRK5 messenger RNA. 20
µg of total RNA from various tissues of rat were separated on 1%
agarose-formaldehyde gel, transferred to nylon membrane, and probed
with P-labeled PCR product (A) and cyclophilin
cDNA (B). The blot was washed at high stringency condition,
followed by autoradiography at -80° C. Samples are thyroid (lane 1), liver (lane 2), heart (lane 3),
kidney (lane 4), colon (lane 5), and lung (lane
6). kb, kilobase(s).
To clarify the functional properties of GRK5 in thyroid cells, especially in the TSH receptor signal transductory system, the cDNA in the sense or antisense orientation (GRK5S or GRK5AS) was stably transfected into rat thyroid FRTL5 cells. After selection with G418, multiple independent cell lines were selected for each transfectant. The data obtained with two clones for each transfectants are shown in Fig. 3and Fig. 4. The FRTL5-GRK5S-1 and -2 expressed approximately 6- and 4-fold higher levels of GRK5 mRNA and 5- and 3-fold higher levels of the protein, respectively, than did the wild type (wt) FRTL5 cells (Fig. 3). In FRTL5-GRK5AS, the expression of the antisense message was detected by RT-PCR with the sense GRK5-specific oligonucleotide primer in the RT reaction (data not shown). FRTL5-GRK5AS-1 and -2 expressed approximately one-forth and one-half of sense GRK5 mRNA and one-third and one-half of the protein, respectively, compared to those in the wt-FRTL5 cells.
Figure 3: Expression of sense or antisense GRK5 messenger RNA in FRTL5 cells stably transfected with sense or antisense GRK5 cDNA. A and B, 15 of µg total RNA from FRTL5 cells stably transfected with the sense or antisense GRK5 cDNA as well as the wt-FRTL5 cells were subjected to Northern blot analysis as described in the legend in Fig. 3(A, GRK5; B, cyclophilin) with an exception that the cDNA probe used in A was prepared by labeling the single stranded pBS containing (+)-strand GRK5 cDNA rescued from the double stranded pBS-GRK5. C, approximately 20 µg of proteins were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF Membrane (Millipore). The blot was incubated with polyclonal rabbit antisera against human GRK5 (Santa Cruz Biochemistry) followed by subsequent incubation with a horseradish peroxidase-conjugated donkey anti-rabbit antibody. Enhanced chemiluminescence detection of GRK5 was performed with the ECL reagents and Hyperfilm-ECL (Amersham). kb, kilobase(s).
Figure 4: Desensitization of the TSH receptor in FRTL5 cells stably expressing sense or antisense GRK5 RNA. The cells cultured without TSH for 7 days were stimulated with 10 units/liter TSH and 0.5 mM 3-isobutyl-1-methylxanthine at 37° C for the indicated periods shown in the horizontal line (A), or the cells were preincubated with 10 units/liter TSH for 4 h, washed extensively, and stimulated with 10 units/liter TSH and 0.5 mM IMBX at 37° C for 30 min (B). Intracellular cAMP concentrations were then measured by radioimmunoassay. Data are representative of three separate experiments; each point represents the mean ± S.D. of triplicate values determined in triplicate cells, and are expressed as % of maximum obtained with 30-min TSH stimulation without TSH pretreatment. *, p < 0.05;**, p < 0.01; NS, not significant (unpaired Student's t test).
In the first series of experiments, these transfectants as well as the wt-FRTL5 cells, cultured in TSH-free medium for 7 days, were incubated with 10 units/liter TSH for up to 24 h. As shown in Fig. 4A, maximum intracellular cAMP concentrations were observed 30 min after TSH stimulation in each cell type. The magnitude of cAMP increase was very similar among three clones (data not shown). In the wt-FRTL5 cells, cAMP levels then gradually declined to 49.3 ± 2.3, 41.0 ± 2.6, and 56.7 ± 3.1% (% maximum, mean ± S.D.) after 2, 4, and 24 h TSH stimulation, respectively. The extent of desensitization was more pronounced in the cells stably expressing the sense GRK5 RNA (FRTL5-GRK5S). For example, cAMP levels 2, 4, and 24 h after TSH stimulation were 26.3 ± 1.5, 13.0 ± 1.0, and 25.3 ± 1.2%, respectively, in FRTL5-GRK5S-1 (p < 0.01 versus the wt-FRTL5 in each point). On the contrary, in cells expressing the antisense GRK5 RNA (FRTL5-GRK5AS), the extent of desensitization after 2, 4, and 24 h incubation with TSH was less evident (for example, 63.3 ± 2.5, 59.7 ± 7.5, and 76.0 ± 6.6% in FRTL5-GRK5AS-1, p < 0.05 versus the wt-FRTL5 in each point).
In the second series of experiments, the wt-FRTL5, FRTL5-GRK5S-1, and FRTL5-GRK5AS-1 were pretreated with 10 units/liter TSH for 4 h, washed extensively, and were then stimulated with 10 units/liter TSH for 30 min (Fig. 4B). The reasons for the 4-h pretreatment of the cells with TSH in these experiments are: (i) the data obtained in the foregoing studies suggested that 4 h incubation of the cells with TSH was sufficient for the maximal desensitization (Fig. 4A), and (ii) previous studies have shown that the TSH receptor down-regulation, another mechanism of the diminished receptor responsiveness, is not manifest after 4 h treatment of FRTL5 cells with TSH(28) . As shown in Fig. 4B, cAMP response to the second TSH stimulation was lower in FRTL5-GRK5S than in the wt-FRTL5 cells (10.3 ± 0.6% of the maximum response in FRTL5-GRK5S versus 28.0 ± 4.6% in the wt-FRTL5, p < 0.01), but higher in FRTL5-GRK5AS (44.7 ± 7.1%, p < 0.05). Furthermore, the basal cAMP level prior to TSH pretreatment was significantly lower in FRTL5-GRK5S cells than in the wt-FRTL5 (1.0 ± 0.1 pmol/well in FRTL5-GRK5S versus 1.3 ± 0.1 pmol/well in the wt-FRTL5, p < 0.05), and that in FRTL5-GRK5AS was slightly, but not significantly, higher than that in the wt-FRTL5 (1.6 ± 0.3 pmol/well, p > 0.05). Differences in the basal levels of cAMP became more evident after 4 h pretreatment with TSH. Thus, those of the wt-FRTL5, FRTL5-GRK5S, and FRTL5-GRK5AS were 8.3 ± 1.5, 2.3 ± 0.6, and 16.3 ± 1.5 pmol/well (p < 0.01), respectively. These data clearly demonstrate that the expression levels of GRK5 was positively correlated with the extent of TSH receptor desensitization.
To evaluate kinase-receptor specificity, another isotype of GRK, GRK6 (10) , was also stably expressed in FRTL5 cells (FRTL5-GRK6S). Two clones, FRTL5-GRK6S-1 and -2, which expressed exogenous GRK6 mRNA (Fig. 5), were selected. In these cells, as shown in Fig. 6, cAMP levels 2 and 4 h after TSH stimulation were significantly lower than those in the wt-FRTL5 cells (for example, 37.2 ± 11.0 and 17.7 ± 7.9% in FRTL5-GRK6S-1 versus 74.2 ± 8.8 and 43.5 ± 10.2% in the wt-FRTL5, p < 0.05 in each point), suggesting that the TSH receptor desensitization can also be influenced by other isotypes of GRKs.
Figure 5: Expression of sense GRK6 messenger RNA in FRTL5 cells stably transfected with sense GRK6 cDNA. A and B, 15 µg of total RNA from FRTL5 cells stably transfected with the sense GRK6 cDNA and the wt-FRTL5 cells were subjected to Northern blot analysis as described in the legend in Fig. 3(A, GRK6; B, cyclophilin). kb, kilobse(s).
Figure 6: Desensitization of the TSH receptor in FRTL5 cells stably expressing sense GRK6 RNA. The cells cultured without TSH for 7 days were stimulated with 10 units/liter TSH and 0.5 mM 3-isobutyl-1-methylxanthine at 37° C for the indicated periods shown in the horizontal line. Intracellular cAMP concentrations were then measured by radioimmunoassay. Data are representative of two separate experiments; each point represents the mean ± S.D. of triplicate values determined in triplicate of cells, and are expressed as % of maximum obtained with 30-min TSH stimulation. *, p < 0.05 (unpaired Student's t test).
In the present studies, we evaluated the molecular mechanisms of homologous desensitization of the TSH receptor in the thyroid. From the extensive studies on desensitization of adrenergic receptors and rhodopsin(2, 3, 4) , we speculated that, like these receptors, phosphorylation of the agonist-bound TSH receptor by GRK(s) and subsequent binding of arrestin(s) to phosphorylated TSH receptor might be involved in receptor desensitization. In the present studies we focused on one of these proteins, GRK, and first attempted to clarify subtypes of GRK expressed in the thyroid by RT-PCR. We also wished to determine if there was a novel thyroid-specific GRK. Although no novel GRK was detected in the thyroid, our data demonstrate that, of 6 different types of GRKs, GRK5 appears to be the predominant isoform expressed in both human and rat thyroids. The fact that little message was detected for GRK4 and GRK6 in Northern blot analysis with rat thyroid RNA (20 µg of total RNA) supports this finding. The expression level of GRK5 mRNA in the thyroid is, however, not higher than that in other tissues such as lung, colon, heart, and kidney in Northern blot analysis.
We next studied the functional properties of
GRK5 in rat FRTL5 thyroid cells by the cloning and transfection of rat
GRK5 cDNA. Enhancement and suppression of GRK5 expression was achieved
by stable transfection of the cloned sense and antisense cDNAs,
respectively. The present studies clearly demonstrate that
overexpression or underexpression of GRK5 in vivo increased or
attenuated, respectively, homologous desensitization of the TSH
receptor. These data, together with the fact that the GRK5 may be the
predominant GRK isoform in the thyroid, implicates GRK5 in TSH receptor
homologous desensitization. Similar approaches have recently been
employed by others and demonstrate in vivo functional
properties of GRKs. Thus, overexpression of -ARK is reported to
enhance
2-adrenergic receptor desensitization(29) , and
suppression of
-ARK expression by antisense oligonucleotides,
overexpression of a
-ARK dominant negative mutant, or neutralizing
antibodies against
-ARK2 successfully attenuates desensitization
of
2-adrenergic, m2 muscarinic receptors, or odorant
receptor(30, 31, 32, 33) .
Furthermore, our data also suggest that GRK5 limits the extent of TSH
receptor homologous desensitization in FRTL5 cells, a finding
consistent with the previous studies with
2-adrenergic
receptor(29) .
The incomplete attenuation of TSH receptor
desensitization in the cells expressing the antisense GRK5 mRNA can be
explained largely by the incomplete suppression of the endogenous GRK5
mRNA expression (Fig. 4A), although another possibility
cannot be excluded that other kinases such as GRK4 and 6, which are
expressed in the thyroid only in a very small quantity, together may
also participate desensitization of the TSH receptor because
exogenously overexpressed GRK6 enhanced desensitization of the TSH
receptor (Fig. 6). Although it is reported that some GRKs prefer
certain types of receptor, the substrate specificity of GRKs is in
general not strict(2, 3) . Indeed GRK5 phosphorylates
rhodopsin, 2- and
2-adrenergic and m2-muscarinic receptors in vitro(4, 9, 34) . Which GRK(s)
are involved in desensitization of the TSH receptor depends not only on
the intrinsic ability of individual GRKs to desensitize the receptor
but also on the concentration of individual GRKs in the thyroid cells.
From these data, we speculate that GRK5 may be capable of
phosphorylating the TSH receptor, although there is at present no
direct evidence supporting this thesis. It has been demonstrated that
phosphorylation of rhodopsin and the 2-adrenergic receptor by GRKs
occurs in the cytoplasmic C-terminal tails(35) , whereas the
phosphorylation site by GRK in the
2-adrenergic receptor appears
to be in the third cytoplasmic loop(36) . We and others have
shown that the truncated TSH receptor in which the
serine/threonine-rich cytoplasmic C-terminal tail of the receptor is
deleted undergoes homologous desensitization, like the full-length
receptor(17, 37, 38) , suggesting other
sites, such as the third cytoplasmic loop of the TSH receptor, may be
targeted by GRKs. Further studies regarding agonist-dependent
phosphorylation of the TSH receptor by GRK5 will be required. This kind
of study could be employed in reconstituted systems with purified
proteins. However, purification of the TSH receptor has not yet been
attained(39) . Furthermore, although it has recently been
reported that crude membranes from insect cells overexpressing GRKs,
rather than purified receptors, can be used for phosphorylation
studies(40) , the functional full-length TSH receptor cannot be
expressed in insect cells. This is probably due to the incomplete or
improper glycosylation of the receptor protein in insect
cells(41, 42) .
In summary, the present studies demonstrate that (i) GRK5 appears to be the predominant isoform of GRK in the thyroid, and (ii) the expression levels of GRK5 are positively correlated with the extent of TSH receptor desensitization. From these data we conclude that GRK5 appears to be involved in homologous desensitization of the TSH receptor in the thyroid.
The nucleotide sequence of rat G protein-coupled receptor kinase
5 has been submitted to the Gen Bank/EMBL
Data Bank with accession number U34841[GenBank].