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Osancor Biotech Inc. (N.F.), Watford, Hertfordshire WD17 3BY, United Kingdom; and Trophogen Inc. (M.W.S.), Rockville, Maryland 20850
Address all correspondence and requests for reprints to: Nadir R. Farid, Osancor Biotech Inc, 31 Woodland Drive, Hertfordshire WD17 3BY, United Kingdom. E-mail: farid{at}osancor96.fsnet.co.uk; or Mariusz W. Szkudlinski, Trophogen, Inc., 6 Taft Court, Suite 150, Rockville, Maryland 20850. E-mail: mszkudlinski{at}trophogen.com.
Abstract
TSH receptor (TSHR) is a member of the leucine-rich repeat-containing G protein-coupled receptors. Both TSHR and its ligand TSH have evolved to acquire specificity, minimize cross-reaction to other glycoprotein hormone receptors, and modulate cognate interaction (and thereby thyrotropic activity). TSHR sequences available from two life orders, teleost and mammals, were analyzed. Teleost TSHRs with low affinity are expressed in many nonthyroidal tissues and show a tendency to gene duplication. In some teleosts, TSHR has limited specificity, and in others extremely high constitutive activity, suggesting the possibility of ligand-independent receptor function. Although mammalian TSHR, in contrast to other glycoprotein hormone receptors, maintains relatively high constitutive activity, the thyrotropic activity of TSH appears to decline in hominoids including humans, probably as part of metabolic adaptation to the changing environment. Critical TSHR residues that determine hormone specificity have been identified in the leucine-rich repeats, and others within the cysteine-rich C-flanking region that determines hormonal activation as well as receptor silencing. Transmembrane (TM) helices, particularly the TM5 and TM6, are likely involved in receptor homodimerization and a unique motif in TM7 appears essential to receptor silencing and internalization. Surprisingly, ternary structures in the intracellular domain as opposed to specific sequence motifs are critical for intracellular TSHR trafficking. It is evident that progress in understanding structure-function relationships of TSHR and its ligand can be further stimulated by inclusion of evolutionary analysis of their primary, secondary and tertiary structure. Such an integrated approach should also contribute to the rational design of highly efficacious therapeutics with either agonistic or antagonistic properties.
THE ADAGE THAT we cannot understand our present and future without referring to the past aptly applies to TSH receptor (TSHR) evolution. And just as in classical paleontology (1), the evolutionary record is less than complete. There is, however, mounting evidence to trace the deep evolutionary history of the TSHR (2). We bring to this review interests focused on receptor and ligand, respectively, to discuss how receptor and ligand have evolved in parallel. This overview is, to a measure, limited by the lack of availability of the cognate ligands for many of the TSHRs cloned and sequenced.
Evolution of TSHR
1) Origins
The TSHR is a member of the GPHR [glycoprotein hormone (GPH) receptor] family, a subset of the large G protein-coupled receptors (GPCRs) superfamily of membrane receptors (2, 3).1 More than 1000 GPCRs have been identified in the human genome, but native ligands have been assigned to only 20%. Many of the GPCRs with unknown ligands display high constitutive activity and at least some may function without a ligand (2, 3), solely based on the regulation of their expression. Although all GPCRs display a certain degree of constitutive activity, the level of such activity varies significantly between various receptors, including GPHRs (5, 6, 7).
The seven-transmembrane (TM) domain apparently evolved to accommodate small ligands between the helices and extracellular loops (3). In LGRs [leucine-rich repeat (LRR) containing GPCRs] of which the glycoprotein receptors are members, a large ectodomain was later incorporated. The ectodomain is composed of many LRRs forming multiple protein-protein interfaces and, assembled sequentially, form a potential ligand-binding surface (Refs. 8 and 9 ; and Figs. 1
and 2, A and B). The two modules, LRR and serpentine, are bridged by the cysteine-rich flanking region (CFR). Variation on the primordial LGR structure resulted from duplication of LRRs, exonic shuffling or recombination, loss of add on sequences (CFLANS) or parts thereof (as in the gonadotropin receptors) (Ref. 2 ; and Fig. 2A). Some LGRs were further selected for specific ligand-receptor interaction, and with enhanced ligand specificity the constitutive activity of the receptor further diminished (5). The association of FSH receptor (FSHR) mutations in the serpentine with spontaneous ovarian hyperstimulation syndrome suggest that a higher constitutive activity of GPHRs may correlate with altered (relaxed) specificity (10, 11)
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2) Coevolution of receptor and hormone
TSHR provided a critical link between pituitary TSH and thyroid function. Although it is not clear when this regulation was first established (in early vertebrates?), the primordial TSHR may have acquired its hormone-dependent function with parallel partial loss of its very high constitutive activity. Further ligand and receptor coevolution (13) resulted in progressive increases in specificity of TSH-TSHR interaction similar to that described for coevolution of gonadotropins and their cognate receptors (Fig. 3). In general, in vitro bioactivities of GPH are closely related to their respective receptor binding activities.
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-subunit of GPH in certain fishes as BBXXXXXBXB (X, any residue; B, basic and can be K-lysine or R-arginine) evolved to the final BXBXXBXXXB basic motif in mammals. It is not accidental that the complete elimination of this motif coincided with the divergence of apes from Old World monkeys. In fact, the intrinsic activity of TSH most likely decreased in apes. However, changes leading to limitation of TSH activity in the evolution of apes are not linked to any nonconservative substitutions modifying receptor function (basal activity, ligand-binding, etc.). Comparisons of hormone and receptor substitution rates as well as of sequences in primates suggest that predominantly conservative substitutions in TSHR contributed to the loss of acidic interface (16, 17). Bovine TSH binds with greater affinity than human (h) TSH to rat and hTSHRs. Although major evolutionary changes in GPHs and their receptors should be considered as ligand-receptor coevolution, the fact is that modification in ligand affinity could evolve in relatively small increments that could be accommodated without major structural changes in the receptor by additional adjustment in expression, internalization, and trafficking (17, 18, 19).
Both functional and structural rationale of basic motif evolution was provided in the follow-up studies on GPHR using the loss-of-superagonist approach. Basic motif in the L1 loop of the -subunit in lower mammals (16, 17), apparently engages Glu 369, Glu 375, and Glu 376 of the human TSHR. The negatively charged patch EEQEDE (357–363) located within CFLANS also contributes to the interaction and explains relatively higher bioactivities of respective superactive analogs with basic motifs in the common hormone -subunit at TSHR than at FSHR and LH receptor (LHR) (Szkudlinski, M. W., unpublished data).
Achieving specificity
The high degree of sequence identity among the GPHs including that within the specific ß-subunits (30–80%), as well as in many domains of their respective receptors, result in glycoprotein hormones capable of interacting with homologous receptors, albeit with low cross-reactivity. At physiological hormone concentrations, this degree of cross-specificity prevents stimulation of homologous receptors. Cross-activation of homologous receptors may, however, be observed at high hormone levels. The resulting clinical manifestations have been termed specificity spillover syndromes (5, 11, 16, 17, 20). Coevolution of TSH and TSHRs were likely directed to control the spillover of TSH activity to homologous gonadotropin receptors and in parallel the thyrotropic activities of gonadotropins at TSHR. Loss and gain of specificity mutagenesis indicated that specificity of TSH-TSHR interactions is achieved by both positive and negative determinants on both ligand and receptor. Highly specific interactions as well as electrostatic repulsion are involved, in accordance with the negative specificity concept. Such negative specificity determinants have been identified in the TSHR LRRs as well as in TSH ß-subunit, within the seat-belt region (11, 17, 21). Studies on this region of the hTSH ß-subunit using chimeric substitutions with gonadotropic sequences suggested that, during the evolutionary diversification of the GPHs from a common ancestral gene (Fig. 3
), determinants of ligand specificity appear to have evolved independently and in a selective fashion. Specifically, replacing the seat-belt domain of the TSH ß-subunit with the corresponding residues of human chorionic gonadotropin (hCG) conferred hCG specificity to the chimera (17).
The GPHs and their receptors diversified as a result of positive selection to adapt to new functions, although little is known about the role of these adaptive mechanisms in sequences diversification between different species. Identification of amino acid substitutions significantly affecting biological activity of the TSH and TSHR support rapid adaptive mechanisms of molecular evolution, as opposed to functionally neutral amino acid replacements resulting from nonselective genetic drift (Refs. 18 and 19 ; and Fig. 4). However, the mere presence of nonconservative amino acid substitution between different species of TSH or TSHR does not predict significant functional difference (e.g. D331G and R63H in TSHR between Old World monkeys and humans). On the other hand, that conservative substitutions may not be trivial is highlighted by the substitution of the highly conserved K183 by R, which changes TSHR specificity (19, 21, 22) (see Footnote 11). In addition to providing specificity, evolution of hormone activity may reflect adaptation of endocrine processes to environmental cues. In this respect, we note that rat (and bovine) TSH have higher intrinsic activity than hTSH, and whereas in rodents and other lower mammals cold exposure is a potent stimulus for TSH secretion participating in thermogenesis, cold is a relatively ineffective stimulus for TSH secretion in man (5). Other more sophisticated mechanisms conserving body heat and promoting thermogenesis have evolved in humans. A more dominant new function for hTSH with attenuated activity and enhanced constitutive activity of hTSHR may be to conserve iodine for thyroid hormone synthesis during periods of nutrient shortages. In this context, similar evolutionary arguments have been advanced for the survival advantage and selection of genes conducive to insulin resistance (23).
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and 5): the LRR TM domains are invariant in length, the CFR shows most variability, and less so the intracellular tail. Fish receptors are characterized by insertions in CFR, exaggerated in the A form of the salmonid receptor with 26 residue duplication with subsequent sequence diversification, whereas single residue deletions (at separate locations) are found in cat and bovine receptors (18, 19) (see Footnote 11). Fish have eight to 10 residue insertions in the intracellular tail and a longer leader peptide in salmonid TSHR. Not much is known about TSHR function in fish, where thyroid tissue is dispersed around the ventral aorta. The cloning and sequencing of seven teleost TSHRs has provided valuable insight into evolving receptor function. Whereas the expression of the two TSHRs cloned from the Amago salmon, is limited to the thyroid, those of sea bass and catfish are found in many other tissues. And whereas both TSH and LH activate sea bass TSHR, catfish TSHR showed much greater selectivity for TSH (18, 24, 25). Based on our assessment, sea bass TSHR has very high constitutive activity (even higher than hTSHR). The regulation of gonadal function may be by modulation of TSHR expression rather than by TSH, which based on protein sequence in several fish species is also lower affinity type. Fish TSHR show hierarchical preference for lower affinity mammalian hormones (18, 24, 25).
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-subunits (29), and its TSHR distinctively has a penultimate extra-exon, supports this notion. Evolutionary Fine Anatomy
Glycosylation sites differ among species
The equivalents of hTSHR glycosylation sites 77, 198, and 302 are preserved in all TSHRs. That at 113 is specific for Homo sapiens and that at 177 for mammals (11, 18, 19, 30). The conclusion that four glycosylation sites, irrespective of location, were necessary for expression and biologic activity (31) is belied by three sites in catfish, salmon-A, and salmon-B TSHR, which are efficiently expressed and stimulated by mammalian TSH (18, 19, 24). Thus, more than just the number of glycan adducts is relevant to receptor trafficking and function. Sea bass, tilapia, and fugu have a distinctive potential glycosylation site (NLT:276–278 in sea bass and 277–279 in tilapia) at the N terminus of CRF (Fig. 5).
Cysteines
The cysteines in the extracellular domain consist of two paired sets at the N and C terminus of the ectodomain, a general orientation typical of LGRs, and an orphan at 176 (hTSHR numbering) (Fig. 4). The S replaced the latter in sheep, and T in teleosts. The former two cysteine sets are thought to be essential for maintaining the secondary structure of the ectodomain; there is, however some uncertainty with respect to the pairing of the C terminus cysteines (5, 18, 30). The equivalent of primate cysteine 24, 29, 31, 41, 283, 284, 301, 390, 398, and 408 are strictly conserved in all TSHRs (5, 18, 30). Cysteines 539 and 599 appear to be strictly primate specific (18). With the exception of the equivalent of C301 in LHR, the cysteines between 383–408 are retained in the gonadotropin receptors.
We have suggested that 390/408 establish covalent linkage, leaving cysteine 398 at the tip of the asymmetric finger. We further suggest that this cysteine finger loops at the base of the CFR to hold in place another finger between cysteines 284 and 301 and through cysteine 398, establish disulfide bond formation with cysteine 283. The cruciate (St. Andrew’s cross) of the intercysteine fingers makes for an efficient restraining influence on TSHR (2)
The LRRs
Modeled on the ribonuclease inhibitor (8), the GPHR have nine LRR motifs arranged as a horseshoe-like structure. Twenty to 25 resides of an LRR comprise a hydrophobic ß-strand, connected by a turn to an -helix. The model predict that the nonleucine (X1–5, Fig. 6) in the ß-strand face outwards, available for interaction with hormone. The details of hormone specificity and specificity switching focused on specific residues in specific LRRs (11, 19, 32, 33, 34, 35, 36). Thus, X5 residue of the ß core of LRR3 and LLR6 were respectively positive and negative determinants in FSHR luteinization. Once bound, LRR4 appears to be important for hormone -subunit association and coupling to the extracellular loops of GPCRs (14, 17, 34). Residues X2, X3, and X4 of the hydrophobic ß-strands of LRR 3 and 9 and X5 in that of LRR7 (Fig. 6) were found to be important for TSHR specificity and switch to LHR/CG receptor (CGR) activity (21). Exchanging 2 and 8 residues, respectively, in FSHR and TSHR for their counterparts in LH/CGR results in receptors with dual specificity. Exchange of a further 12 residues is necessary for TSHR to lose its thyrotropic attributes. The changes in specificity are associated with changes in surface electrostatic charges in different patches of the horseshoe (11, 21)
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and 6). Except for LRR1 and LRR9, none of the rate-shift sites are involved in the hydrophobic ß-strand, facing the ligand in the horseshoe conformation of LRR (8, 9). There are, however, interesting rate shifts in sites proximal to LRR ß-strands e.g. that at residue 103, just proximal of the TSHR-specific motif in LRR3, THIERN, which forms an integral part of the LRR3 hydrophobic ß-strand (Figs. 4 and 5). Rate-shift sites in LRR1 and LRR 9 involve, respectively, residues 59 and 60, and 253 and 255. The latter two LRR9 residues (and their counterparts in LRR3) form part of the TSH-binding pocket (246–260), made up of three discontinuous domains (37). Residue 255 is R in all vertebrates but is K in rat, and although the substitution is conservative, K is more soluble than R. The substitutions at 253 are more radical: I in mammals and salmon and K in sea bass and fugu but S in catfish, substitutions that would likely impact receptor thyrotrophic characteristics.
The lack of rate-shift sites in the central LRRs (2, 3, 5, 6, 7, 8) (see Footnote 11) speaks strongly for the essential role of the hydrophobic ß-strands in TSHR function despite the changing features of ligand and other parts of the receptor more than 400 My of evolution (Fig. 5).
The K183R mutation results in human TSHR luteinization (17, 22). This residue is conserved in the LHRs and falls within LRR5 in a region in which domain-exchange experiments between the two receptors suggested a switch in specificity. K183 is preserved in all mammals, but its equivalent position is substituted for M in teleosts. This substitution would result in TSHR luteinization (18, 19, 22). K183 forms a salt bridge with E157, a residue conserved in all LGRs and mutation of the former to R results in the disruption of the salt bridge and establishment of an association with a distal D232; change to M would likely result in the establishment of alternative partners (22). These mutations clearly cause changes in the ternary structure of adjacent LRR and electrostatic potential to allow GC to be a biologic ligand at TSHR.
That residue 113 was highlighted as rate-shift site emphasizes the sensitivity of the method because it is a Homo sapiens-specific site of N-linked glycosylation of TSHR (19).
The second domain of the TSH binding pocket, 277–296, is absolutely conserved in all 17 TSHRs as well in the other GPHRs (18, 19) (see Footnote 11) and is a domain previously identified as being important in LHR/CGR activation but not binding (38, 39).
The CFR region
The LRR segment of the ectodomain is important, both as a conduit and as a dynamic modulatory and modulated participant in TSH binding and initiation of receptor activation. Once anchored to the CFR, TSH initiates changes in receptor ternary structure, including oxidation of the disulfide bonds that precede receptor cleavage, reoxidation of the disulfide bonds to create covalently bound dimers between stumps of the remainder of the ectodomain (2, 18, 40, 41, 42) to enhance signaling. Several lines of evidence suggest TSH -subunit is involved in signaling, whereas the ß-subunit is involved in hormone specificity (5, 21, 34, 39).
The replacement of serine 281 immediately preceding the HCCAF motif (Figs. 2B and 5), an innovation in the GPHRs (2, 18, 38) by residues of different volume or charge results in constitutive action of TSHR (38), as is seen in the sea bass. Serine 281 imposes a silencing influence on the receptor (18, 43) and mutation of the C283 in the HCCAF motif also causes constitutive activation by disrupting ternary structure (12, 18).
Mammalian CFR is predicted to comprise four -helices forming a compact structure stabilized by noncovalent force interactions, with most of CFLANS projecting out between the second and third helices. CFLANS random coil structure and hydrophilicity allow easy access to the cleaving enzyme and immunocompetent cells (2).
Primates have a distinctive 306–307 methionine glutamine (MQ) doublet immediately upstream from site I for TSHR cleavage (18, 30), and given the role that has been postulated for CFR cleavage in the pathogenesis of Graves’ disease (44, 45), it is possible that Old World monkeys’ receptors are cleavable. There is, however, no information that they develop spontaneous autoimmune hyperthyroidism. We have identified a disintegrin and metalloprotease 10 as the sheddase responsible for hTSHR cleavage. It digests membrane-anchored structures at defined distances from the membrane, has recognition motifs, although there is less certainty as to whether they have consensus cleavage sequences (46). Contrary to previous studies (30), it is apparent when the ectodomain is foreshortened at the N terminus (314–317) or C terminus (367–369) CFLANS, the balance of the receptor is constitutively activated and its capacity for internalization and cycling are altered (47). We suggest that CFLANS may contribute to TSHR’s constitutive basal activity (compared with FSHR and LHR). Although not rigorously tested, the silencing effects of selected regions of the ectodomain, including the CFR, on the constitutive activity of the serpentine could be magnified by the absence of CFLANS (7), reminiscent of constitutive activities of teleost TSHRs.
CFR sports 14 rate-shift sites. No rate-shift residues are implicated for the distal site of ectodomain digestion, but residue 317 shows trans-species polymorphism (L/M) (Fig. 5). The relevance of this finding is unclear because TSHR cleavage is generally assumed to be hTSHR specific, although this issue has not been specifically addressed in other species (18, 19). The R310C mutation results in a TSHR that is constitutively activated but incapable of binding TSH (48) (see Footnote 11), whereas the mutation at 324 is a nonsense mutation (see Footnote 11). The 310 substitutions (R in mammals) show divergence in fish: Q in salmon, H is sea bass, L in fugu, and D in catfish. These substitutions are likely to influence receptor ligand binding and constitutive activity. The R310C mutation may result in local disruption of the second -helix and in loss of the helix-helix interaction maintained by noncovalent forces. At the equivalent position, FSHR shares an R with TSHR, which is substituted by K in LHR; it is unclear to what measure this conservative substitution influences LHR function. A motif that occurs immediately adjacent to the membrane (FNPCEDIMGY) and that is identical between LH/CGR and TSHR has been described in the former to be important for hormone activation but not binding (49). The third epitope/domain making up the TSH binding pocket, FDSHY residues 381–385, is distal to the TSHR signature motif LKNPQE, both C-terminal to the 50 residue insertion. A rate-shift site (residue 382) centers this domain (37) and with the introduction of negatively charged apartate (Q vs. D in mammals) would impact TSH action and likely receptor immunogenecity (18, 19) in fish (Fig. 5). Sulfation of Y 385 is essential for cognate agonist recognition by TSHRs and indeed by all mammalian GPHRs (50, 51). It is apparent that the ternary structure of CFR involves at least one salt bridge in that the mutagenesis of either E or D in the proline cysteine glutamic asparagine (PCED) motif disrupts in a salt concentration-dependent manner TSHR ligand binding and signaling (52) and the sulfated Y385 may establish a salt bridge with TSH -subunit (17). The juxtamembrane part of the ectodomain maintains LRR domain helicity (53).
The serpentine
The modeling of the serpentine portion of GPHR on the rhodopisn receptor, albeit in the inactive conformation (54), has rationalized the influence of natural and introduced mutations as well as that of ligand engagement on the ternary TSHR structures (11, 55). Dimerization of TSHR, long entertained on the basis of indirect evidence, was recently supported by direct observations (reviewed in Ref. 18). There is also evidence for high order oligomers. After TSHR cleavage, homodimerization ensues and involves covalent bond formation between the residual ectodomain stumps. Contact or domain-swapped dimers occur between the external aspects of helices 5 and 6 (and to a lesser measure 1 and 7) of TSHR, whereas other helices (2, 3, 4) may be involved in high-order oligomerization (18). TSHR mutations that modify receptor function occur in residues that are either strictly conserved or conservatively substituted in all TSHR sequences and all were absolutely conserved in mammals (18, 55).
TM6 exhibits a stretch of five residues (629–633), part of the mutation cluster region (55), each of which when mutated results in constitutively activated receptor. The wild-type conformation of this mid-region (
1.5 turn of the -helix) silence the receptor. D633 and T632 part of a motif [phenylalanine threonine asparagine (FTD)] highly conserved in GPHRs silences the receptor by a complex interaction with N 674 (of NPXXY motif) at the cytoplasmic end of helix TM7, an interaction that is broken by gain of function mutations (11, 56). The mutation cluster region falls within a region (the equivalent of TSHR 618–635) identified as a potential dimerization domain in ß2-adrenergic receptor (57) and which is highly conserved in all LGRs. This region maintains similar spacing of charged and hydrophobic residues throughout the rhodopsin subfamily of GPCRs, suggesting its potential importance in homo- and heterodimerization of various receptors.
Rate-shift sites were detected in all TM helices except 6 and in the third intracellular loop. Residue 425 in TM1, a site of mutation (see Footnote 11), is placed on the external face of the helix, as are two of the rate-shift residues (18, 19). In rat TSHR, P and M, respectively, replace L in 426 and 427 found in all other receptors (Fig. 4), with likely important consequences in helix packing. Residue 438 shows an interesting cross-species polymorphism. Residue 495 at the beginning of TM3 is next to C494, a site of activating TSHR mutations (see Footnote 11), and the variety of substitutions in 495 are likely to influence receptor function. Likewise, the cluster of rate-shift residues 540, 543, and 547 surround a site (W546), conserved in all GPCRs, and associated with loss of TSH response when mutated (see Footnote 11). These rate-shift residues would likely influence TM helicity and charge (18, 19). Residue 600 mutation is associated with TSH unresponsiveness, and Y601 is important for human receptor constitutive basal activity and receptor coupling to Gq/11 (16, 18, 19). The placement and nature (F in sea bass and fugu as opposed to alphitic residues in other vertebrates) of rate shift at residues at 602 at the cytoplasmic end of helix 5 will undoubtedly influence receptor/G protein coupling.
Rate-shift residues found in helices 1, 3, and 5 map to the outer faces of the helices. Those in helix 1 and 5 are likely relevant to receptor function because outside residues are involved in the dimerization of TM5 and 6 and TM1 and 7 (18) (see Footnote 11).
The third intracellular loop and the juxtamembrane portion of TM6 are crucial to the engagement with Gs and its activation (4, 58). The substitutions at 606 in the third intracellular loop are relatively conservative except (Y) in catfish and the rate-shift position 614 almost certainly influences the quality and perhaps intensity of the transduced signal (5, 6) (see Footnote 11). A deletion (613–621) results in the activation of TSHR, as does a point mutation at 619. D619 apparently forms an ionic lock with the canonical glutamic arginine tryptophan (ERW) motif at the bottom of TM3 (11). Both Caenorhabditis elegans and Fly 1 LGRs are constitutively active (59, 60), in the former related to alteration in the ERW motif (59). Fly 1 LGR constitutive activity is further enhanced by introducing activating mutations equivalent to TSHR 619 or 633 (60). In teleosts, 614 shows a remarkable degree of polymorphism. The polar N in mammals is substituted by the nonpolar C in salmon-B and with the positively charged R and H in sea bass and fugu and again with polar T in catfish (Fig. 5).
The highly conserved motif (NPXXY, 674–678) at the end of TM7 not only important in receptor silencing but is also essential for TSHR coupling to G protein as well as internalization (56, 61).
The cytoplasmic tail
The high degree of sequence variability even within the same order of life and thus the high number of rate shifts (18) in the intracellular tail may reflect the nonessential role of some residues involved in rate shifts, the importance of nonlinear domains, diversification of receptor signaling, or combinations thereof. That the deletion of the C terminus of this part of the receptor and mutation of several potential phosphorylation sites did not compromise receptor function or internalization (62) focuses in on the role of the tail in receptor recycling (63, 64).
Apparently, endosome degradation of internalized GPCR is the default trafficking mechanism (65). A number of motifs in the intracellular tail have been identified in a number of GPCR, including LHR, which along with covalent modifications in some receptors permit recycling (66, 67, 68). Cytoplasmic tail addressograms exist in TSHR (69) but are not identifiable by comparison to those in other GPCRs (66, 67, 68), and may well depend on discrete residues presented for covalent modification or coupling with partner protein by ternary and possibly dynamic conformation of the tail (70). Most motifs include an L, which appears to be substituted in teleost as opposed to mammalian receptors.
Perhaps not surprisingly, residue 727 polymorphic in hTSHR is a site of rate shift that is faster in mammals compared with fish (Ref. 5 ; and Fig. 4). Although the D727E polymorphism in humans does not appear to modify wild-type TSHR function, it has a silencing influence on a constitutively activated receptor (71). This observation raises the possibility that some rate-shift sites in hinge region or TM domain may be compensated for by changes in other TM regions or indeed the intracellular tail.
To further study functional relevance of evolutionary changes in TSH and TSHR, we envision generation of transgenic animals with selected simultaneous replacements in both TSH and TSHRs to test their role and contribution to specific physiological, metabolic, and environmental adaptations.
Acknowledgments
N.R.F. acknowledges the valuable contributions made by several collaborators to this work including Bjarne Knudsen, Viktoria Kaczur, Lászlò Puskás, and Yufei Shi. M.W.S. thanks Bruce Weintraub for continuous support and stimulating discussions on the evolution of TSH activity.
Footnotes
Abbreviations: CFLANS, CFR add on sequences; CFR, cysteine-rich flanking region; CGR, CG receptor; FSHR, FSH receptor; GPCRs, G protein-coupled receptors; GPH, glycoprotein hormone; GPHRs, GPH receptors; hCG, human chorionic gonadotropin; hTSHR, human TSHR; LGRs, LRR containing G protein-coupled receptors; LHR, LH receptor; LRRs, leucine-rich repeats; TM, transmembrane; TSHR, TSH receptor.
1 See this web site: http://www.gpcr.org. 
Received April 7, 2004.
Accepted for publication June 25, 2004.
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2-based test. At each position in the alignment, the method performs the likelihood ratio test for the significance of a rate shift at the separation point between mammals and fish. The simulated distribution was found using 10,000 replicates, ensuring an accurately determined 5% cut-off for significance. The colored sites are the significant rate-shift sites, with red and blue indicating fast and slow evolution, respectively. There were 82 rate-shift sites, significantly more frequent than predicted (38.2, P < 0.0001). There were 52 sites in mammalian TSHRs where the rates were significantly faster than in fish. The rate-shift frequency is not necessarily higher in domains known for structural plasticity in mammalian TSHRs (
S in pig, 3 P