- Email: [email protected]
peptide YY receptor Y1 cDNA from Xenopus laevis
BB,
Biochi~ic~a et B~bphysica A~ta ELSEVIER
Biochimica et Biophysica Acta 1261 (1995) 439-441
Cloning and sequence analysis of a neuropeptide Y/peptide YY receptor Y1 cDNA from Xenopus laevis Anders G. Blomqvist
a,* Eric W. Roubos b, Dan Larhammar a Gerard J.M. Martens b
a Department of Medical Genetics, Uppsala University, Box 589, S-751 23 Uppsala, Sweden b Department of Animal Physiology, University. of Nijmegen, P.O. Box 9010, 6500 GL Nijmegen, The Netherlands Received 6 September 1994; revised 21 February 1995; accepted 22 February 1995
Abstract
Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that share at least two distinct receptors denoted Y1 and Y2. The Y1 receptor has previously been cloned in man, rat and mouse• We describe here the cloning and sequence of a Xenopus laevis Y1 receptor that shares 81% amino acid sequence identity with the human receptor in the region spanning transmembrane (TM) regions I to VII. The extracellular amino-terminal part, TM IV and the second extracellular loop contain several replacements suggesting that these portions have no or limited direct interactions with the peptide ligands. The intracellular regions including the carboxy-terminal tail are nearly identical between Xenopusand mammals, suggesting strong structural constraints on the portions that may interact with G proteins• Keywords: Neuropeptide Y; Peptide YY; Receptor; Evolution; Muscarinic; Mutagenesis; (X. laevis)
Neuropeptide Y (NPY) is a widely and abundantly expressed peptide in the nervous system of all vertebrates investigated• Its structure has been highly conserved during vertebrate evolution [1]. NPY is 36 amino acids long and belongs to a family that includes peptide YY (PYY) and pancreatic polypeptide (PP). Both NPY and PYY can bind to two distinct receptors named Y1 and Y2 [2]. A third type of receptor, Y3, seems to be more NPY specific and does not bind PYY [2]. PP binds to neither of these receptors. Instead there are data which indicate the existence of a unique PP receptor [3]. The YI receptor has recently been cloned in human [4,5], rat [6,7] and mouse [8]. It is a typical 7TM (seven transmembrane regions) receptor that couples to G proteins and inhibits cAMP production. The clone proposed to encode a bovine Y3 receptor [9] has been found not to bind NPY or PYY when it is expressed in eukaryotic cells [10,11]. Recently, the angiotensin II type 1 receptor was cloned in Xenopus laevis [12] and found to differ from mammalian receptors by having a lower affinity to non-peptide The nucleotide sequence data reported in this paper have been deposited to the EMBL/GenBank Data Libraries under the accession No. L25416. * Corresponding author• Fax: + 46 18 526849. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 0 5 1 - 8
ligands [13] Also other receptors have been found to differ between species in pharmacological properties (tachykinin receptors, 5HT1B-5HT1D/3 receptors)• Thus, species comparisons may help identify structurally important positions in the receptors, not only for binding of pharmacological substances but also for endogenous ligands and G proteins. Therefore, we decided to compare the structures of vertebrate Y1 receptors. We describe here the Xenopus laevis Y1 receptor because this species is commonly studied in several branches of biology. In particular, NPY has received much attention because of its important role in the control of background adaptation in Xenopus laevis [14,15]. The sequence of Xenopus laevis NPY was recently elucidated by cDNA cloning [16,17]. In order to isolate a cDNA encoding a Xenopus Yl-receptor we screened a Xenopus laevis hypothalamus cDNA library (hZAPII) under low-stringency conditions (25% formamide at 37 ° C) with a 855 bp PCR-derived probe spanning amino acids 44-329 of the human Y1 receptor. Three hybridization-positive clones were obtained and rescued according to the manufacturer's protocol (Stratagene, La Jolla, CA). Deletion subclones were generated with suitable restriction enzymes. Clones were sequenced by employing Sanger's dideoxy chain termination procedure using an automated fluorescent dye DNA sequencer instrument (Applied Biosystems) or [ 3 3 p ] d A T P and [ 3 5 5 ] d A T P
A.G. Blomquist et al. / Biochimica et Biophysica Acta 1261 (1995) 439-441
440
(Amersham) followed by autoradiography. Additional oligonucleotide primers were synthesized to resolve ambiguities in the sequence. DNA sequences were analyzed by using SeqEd vl.0.3 (Applied Biosystems). One full-length clone (XYI-1, 2.1 kb insert)was completely sequenced. The Y1 receptor is 81% identical between Xenopus and human in the segment spanning TMI-TMVII (Fig. 1). In comparison, the two Xenopus laevis angiotensin II type 1 (AT 1) receptors are only 67% and 68% identical to the human AT 1 receptor in the corresponding region. One additional 7TM receptor is known in Xenopus, the dopamine D2 receptor [18] This receptor has even higher identity between Xenopus and human than the Y1 receptor, but is more divergent in parts of the large third intracellular loop. Most of the differences between the Xenopus and human Y1 receptors reside in the amino-terminal extracellular segment, transmembrane region IV and extracellular loop 2 (Fig. 1). The differences suggest that these parts of the receptor have no or only minor direct interactions with the ligands. In contrast, most other parts of the receptor are highly conserved, and the replacements that have occurred are usually of conservative nature even in the loops. This suggests that there is strong conservative selection pressure on these parts of the receptor, probably both for ligand-binding residues and residues that are involved in the conformation of the transmembrane regions and G-protein interactions. Recently, Walker and colleagues [19] identified by site-directed mutagenesis several aspartate residues in the extracellular loops of the human Y1 receptor that seem to be important for ligand binding. Complete loss of NPY binding was observed for the mutations D194A (D194 mutated to A), D200A, D205A and D287A. These mutations correspond to XY1 189, 195 and 200 in extracellular I
I
I
loop 3 and 283 in extracellular loop 4. It was hypothesized that at these positions salt bridges are formed with human NPY positions Arg-19, Arg-25, Arg-33, and Arg-35. Xenopus NPY differs from human NPY at position Arg-19, and has instead Lys (which is probably the ancestral amino acid; see Ref. [20]). This position was suggested to interact with Asp194 in the human Y1 receptor. In Xenopus this position (189) has Gly, a neutral amino acid. Thus, no salt bridge can be formed between these two amino acids in Xenopus. Possibly the absence of Asp at position 189 (in Xenopus) may be compensated for by either or both of two other negative residues in the same loop at positions 196 (Asp) and 199 (Glu). Additional studies are required to confirm the proposed ligand-receptor interactions, for instance by changing the other three Asp residues in the human Y1 receptor to Glu to maintain the negative charge and by replacing Xenopus Y1 positions 196 and 199. Furthermore, it is interesting to note that the less conserved amino terminus has two conserved acidic residues at positions Glu-10 (Xenopus pos. 8) and Asp-29 (Xenopus pos. 27). However, as neither of these positions affected binding when mutated to Ala [19] there clearly can be other reasons for maintaining extracellular charged residues than interaction with ligands. A conserved aromatic residue in the third intracellular loop is interesting with respect to the work by Bliiml and colleagues [21,22]. Their study on the rat muscarinic receptor M3 shows that a Tyr-residue (bold and underlined) in the sequence YWRIYKETEKRT, is important in G-protein coupling to the PI pathway. Site-directed mutagenesis has shown that the M3 receptor requires an aromatic residue, Tyr, Phe or Trp, in this position to get efficient PI coupling. It has been suggested that this is a general recognition motif for G-proteins [23,24] since various Gs, Gq and Gi-coupled receptors have this aromatic residue. Also the
I
I I f I 1 I I TM I TM I I MAN: MN~STLFSQVENHS-VHSNFSEKNAQLLAFEND~CHLpLAMIFTLALAY.GAVIILG~SGNLALIIIILKQKEMRNVTNILIVNLSFSDLLVAIMCL~FTFv RAT: ....7.R Y Y V N-SPF V ~ V MOU: ..;_ K I Y A N-SP V ~ V XEN: _f.._ Y - - L PN I GNITFPI--SE - A P L ~ AT LI
aa 99 98 98 94
TM I I I TM IV MAN: YT~MDHwVFGEAMCKLNPFVQCvSITvSIFSLvLIAVERHQLIINPRG~RPNNRHAYVGIAvIWvLAVASSLPFLIY~VMTDEPFQNvTLDAYKDKYvCF RAT: T I T V IL S A F MOU: T I T V IL S A F XEN: I V EYI V I CF T GF M C T LMM S L K IS S IG L
199 198 198 194
TM V TM V l MAN: DQFPSDSHRLSYTTLLLVLQYFGPLCFIFICYFKIYIRLKRRNNMMDKMRDNKYRSSETKRINIMLLSIvvAFAVC•LPLTIFNTvFD•NHQIIATCNHN RAT: K I S V MOU: K I S XEN: ED E KF FI L V T FL I G L FF EAV
299 298 298 294
TM V I I MAN : LLFLLCHLTAM•STC•NP•FYGFLNKNFQRDLQ•FFNFCDFRSRDDDYET•A•STMHT•V•KTSLKQASPVAFKKINNNDDNEK•* RAT: SM MOU: SM XEN: I E I *
* V*
384 382
382 366
Fig. 1. Alignment of NPY receptors from man, rat, mouse, and Xenopus. The human Y1 receptor shares 81% identity to the Xenopus Y1 receptor in the region spanning TMI-TMVII. The amino-terminal and the carboxy-terminal parts of these receptors share 47% and 96% identity, respectively. Dotted underlinings indicate putative glycosylation sites. * denotes termination.
441
A.G. Blomqvist et al. /Biochimica et Biophysica Acta 1261 (1995) 439-441
Xenopus Yl-receptor (YTKIFLRLKRRN) and the three mammalian Yl-receptors (YF-KIYIRLKRRN) have an aromatic residue in this position. Another conserved sequence of the Y1 receptor family is the ERH motif (ERItQLIIN) in the beginning of the second intracellular loop. In other peptide receptors this sequence is usually DRY or alternatively DRH, DRF, DRC, ERY, or ERW. Thus, the ERH motif seems to be unique for Yl-receptors. It will be interesting to see whether other NPY/PYY-receptors subtypes also have the ERH motif. The cloned Xenopus laevis Y1 receptor has been cloned in different expression vectors (pTEJ8, pcDNAI/Neo, pCD-PS) where the (GA)~s repeat in the 5'-untranslated region of the cDNA has been deleted, as this segment may compromise the expression efficiency. Nevertheless, the level of expression has been too low to give satisfying levels of expressed protein for binding studies. The human Y1 receptor has also been found to be difficult to express when transfected transiently in contrast to the rat Y1 receptor, that works sufficiently well. Sequence comparisons reveal that the sequence motif 'ATIq'A' is present in the Xenopus sequence five times, in the human receptor sequence four times, and in the mouse and rat receptor sequences only once. This motif has been shown to be a target for RNA-destabilizing enzymes and may therefore rapidly decrease the half-life of the mRNA [25]. An example of a cDNA with motifs for mRNA degradation within the coding regions is c-fos [25,26]. Another possibility is the presence of hairpin loops in the mRNA that decrease the translation efficiency. A third explanation might be that the cloned Xenopus Y1 receptor is non-functional, because Xenopus laevis has a tetraploid genome and numerous loci are present in duplicate. However, for another Xenopus receptors that exists in duplicate, both copies have been found to be functional [12]. Furthermore, it would seem unlikely that a 366 amino acids long sequence would not contain any frameshift- a n d / o r nonsense mutations if it does not have functional role. Subclones of the XYI-1 clone are currently used for in situ hybridization to study the cellular localization and level of expression of the Y1 receptor in the brain and pituitary of Xenopus laevis during background adaptation of the animal. This is of physiological significance because NPY is an important regulator of the adaptation process and it has recently been shown that the amounts of NPY peptide and mRNA in neurons of the suprachiasmatic nucleus differ greatly between black- and white-adapted animals [27]. We are grateful to Ann-Sofie EngstriSm, Charlotte Rouppe van der Voort and Maarten van Riel for their contributions to this work. This work has been supported by grants from the Swedish Natural Science Research Council (B-BU 8524-320), from the Netherlands Organization for Scientific Research (NWO), and from the Euro-
pean Community (BIOT-91-0302 BCHRXCT 920017).
and
HCM
ER-
References [1] Larhammar, D., S6derberg, C. and Blomqvist, A.G. (1993) In The Biology of Neuropeptide Y and Related Peptides (Colmers, W.F. and Wahlestedt, C., eds.), pp. 1-42, Humana Press. [2] Grundemar, L., Sheikh, S.P. and Wahlestedt, C. (1993) In The Biology of Neuropeptide Y and Related Pepfides (Colmers, W.F. and Wahlestedt, C., eds.), pp. 197-240, Humana Press. [3] Schwartz, T.W., Sheikh, S.P. and O'Hare, M.M.T. (1987) FEBS Lett. 225, 209-214. [4] Herzog, H., Hort, Y.J., Ball, H.J., Hayes, G., Shine, J. and Selbie, LA. (1992) Proc. Natl. Acad. Sci. USA 89, 5794-5798. [5] Larhammar, D., Blomqvist, A.G., Yee, F., Jazin, E., Yoo, H. and Wahlestedt, C. (1992) J. Biol. Chem. 267, 10935-10938. [6] Krause, J., Eva, C., Seeburg, P.H. and Sprengel, R. (1992) Mol. Pharmacol. 41, 817-821. [7] Eva, C., Kein~inen, K., Monyer, H., Seeburg, P. and Sprengel, R. (1990) FEBS Lett. 271, 81-84. [8] Eva, C., Oberto, A., Sprengel, R. and Genazzani, E. (1992) FEBS Lett. 314, 285-288. [9] Rimland, J., Xin, W., Sweetnam, P., Saijoh, K., Nestler, E.J. and Duman, R.S. (1991) Mol. Pharmacol. 40, 869-875. [10] Jazin, E.E., Yoo, H., Blomqvist, A.G., Yee, F., Weng, G., Walker, M.W., Salon, L, Larhammar, D. and Wahlestedt, C. (1993) Regul. Pept. 47, 247-258. [11] Herzog, H., Hort, Y.J., Shine, J. and Selbie, L.A. (1993) DNA Cell Biol. 12, 465-471. [12] Bergsma, D.J., Ellis, C., Nuthulaganti, P.R., Nambi, P., Scaife, K., Kumar, C. and Ayiar, N. (1993) Mol. Pharmacol. 44, 277-284. [13] Ji, H., Sandberg, K., Zhang, Y. and Catt, K.J. (1993) Biochem. Biophys. Res. Commun. 194, 756-762. [14] Jenks, B.G., Leenders, H.J., Martens, G.J.M. and Roubos, E.W. (1993) Zool. Sci. 10, 1-11. [15] Roubos, E.W., Martens, G.J.M. and Jenks, B~G. (1993) Ann. N.Y. Acad. Sci. 680, 130-134. [16] Van Riel, M.C.H.M., Tuinhof, R., Roubos; E.W. and Martens, G.J.M. (1993) Biochem. Biophys. Res. Commun. 190, 948-951. [17] Griffin, D., Minth, C.D. and Taylor, W.L. (1994) Mol. Cell Endocrinol. 101, 1-10. [18] Martens, G.J.M., Molhuizen, H.O.F., Gr6neveld, D. and Roubos, E.W. (1991) FEBS Lett. 281, 85-89. [19] Walker, P., Munoz, M., Martinez, R. and Peitsch, M.C. (1994) J. Biol. Chem. 269, 1-7. [20] Larhammar, D., Blomqvist, A.G. and S6derberg, C. (1993) Comp. Biochem. Physiol. 106, 743-752. [21] Bliiml, K., Mutschler, E. and Wess, J. (1994) J. Biol. Chem. 269, 11537-11541. [22] Bliiml, K., Mutschler, E. and Wess, J. (1994) J. Biol. Chem. 269, 402-405. [23] Arden, J.R., Nagata, O., Shockley, M.S., Philip, M., Lameh, J. and Sadee, W. (1992) Biochem. Biophys. Res. Commun. 188, 11111115. [24] Cheung, A.H., Huang, R.C. and Strader, C.D: (1992) Mol. Pharmacol. 41, 1061-1065. [25] Belasco, J. and Bawerman, G. (1993) Control of messenger RNA stability, Academic Press, New York. [26] Shyu, A.-B., Greenberg, M.E. and Belasco, LG. (1988) Genes Dev. 3, 60-72. [27] Tuinhof, R., Laurent, F.Y.S.C., Ebbers, R.G.E., Smeets, W.J.A.J., Van Riel, M.C.H.M. and Roubos, E.W. (1993) Neuroscience 55, 667-675.
Biochi~ic~a et B~bphysica A~ta ELSEVIER
Biochimica et Biophysica Acta 1261 (1995) 439-441
Cloning and sequence analysis of a neuropeptide Y/peptide YY receptor Y1 cDNA from Xenopus laevis Anders G. Blomqvist
a,* Eric W. Roubos b, Dan Larhammar a Gerard J.M. Martens b
a Department of Medical Genetics, Uppsala University, Box 589, S-751 23 Uppsala, Sweden b Department of Animal Physiology, University. of Nijmegen, P.O. Box 9010, 6500 GL Nijmegen, The Netherlands Received 6 September 1994; revised 21 February 1995; accepted 22 February 1995
Abstract
Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that share at least two distinct receptors denoted Y1 and Y2. The Y1 receptor has previously been cloned in man, rat and mouse• We describe here the cloning and sequence of a Xenopus laevis Y1 receptor that shares 81% amino acid sequence identity with the human receptor in the region spanning transmembrane (TM) regions I to VII. The extracellular amino-terminal part, TM IV and the second extracellular loop contain several replacements suggesting that these portions have no or limited direct interactions with the peptide ligands. The intracellular regions including the carboxy-terminal tail are nearly identical between Xenopusand mammals, suggesting strong structural constraints on the portions that may interact with G proteins• Keywords: Neuropeptide Y; Peptide YY; Receptor; Evolution; Muscarinic; Mutagenesis; (X. laevis)
Neuropeptide Y (NPY) is a widely and abundantly expressed peptide in the nervous system of all vertebrates investigated• Its structure has been highly conserved during vertebrate evolution [1]. NPY is 36 amino acids long and belongs to a family that includes peptide YY (PYY) and pancreatic polypeptide (PP). Both NPY and PYY can bind to two distinct receptors named Y1 and Y2 [2]. A third type of receptor, Y3, seems to be more NPY specific and does not bind PYY [2]. PP binds to neither of these receptors. Instead there are data which indicate the existence of a unique PP receptor [3]. The YI receptor has recently been cloned in human [4,5], rat [6,7] and mouse [8]. It is a typical 7TM (seven transmembrane regions) receptor that couples to G proteins and inhibits cAMP production. The clone proposed to encode a bovine Y3 receptor [9] has been found not to bind NPY or PYY when it is expressed in eukaryotic cells [10,11]. Recently, the angiotensin II type 1 receptor was cloned in Xenopus laevis [12] and found to differ from mammalian receptors by having a lower affinity to non-peptide The nucleotide sequence data reported in this paper have been deposited to the EMBL/GenBank Data Libraries under the accession No. L25416. * Corresponding author• Fax: + 46 18 526849. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 0 5 1 - 8
ligands [13] Also other receptors have been found to differ between species in pharmacological properties (tachykinin receptors, 5HT1B-5HT1D/3 receptors)• Thus, species comparisons may help identify structurally important positions in the receptors, not only for binding of pharmacological substances but also for endogenous ligands and G proteins. Therefore, we decided to compare the structures of vertebrate Y1 receptors. We describe here the Xenopus laevis Y1 receptor because this species is commonly studied in several branches of biology. In particular, NPY has received much attention because of its important role in the control of background adaptation in Xenopus laevis [14,15]. The sequence of Xenopus laevis NPY was recently elucidated by cDNA cloning [16,17]. In order to isolate a cDNA encoding a Xenopus Yl-receptor we screened a Xenopus laevis hypothalamus cDNA library (hZAPII) under low-stringency conditions (25% formamide at 37 ° C) with a 855 bp PCR-derived probe spanning amino acids 44-329 of the human Y1 receptor. Three hybridization-positive clones were obtained and rescued according to the manufacturer's protocol (Stratagene, La Jolla, CA). Deletion subclones were generated with suitable restriction enzymes. Clones were sequenced by employing Sanger's dideoxy chain termination procedure using an automated fluorescent dye DNA sequencer instrument (Applied Biosystems) or [ 3 3 p ] d A T P and [ 3 5 5 ] d A T P
A.G. Blomquist et al. / Biochimica et Biophysica Acta 1261 (1995) 439-441
440
(Amersham) followed by autoradiography. Additional oligonucleotide primers were synthesized to resolve ambiguities in the sequence. DNA sequences were analyzed by using SeqEd vl.0.3 (Applied Biosystems). One full-length clone (XYI-1, 2.1 kb insert)was completely sequenced. The Y1 receptor is 81% identical between Xenopus and human in the segment spanning TMI-TMVII (Fig. 1). In comparison, the two Xenopus laevis angiotensin II type 1 (AT 1) receptors are only 67% and 68% identical to the human AT 1 receptor in the corresponding region. One additional 7TM receptor is known in Xenopus, the dopamine D2 receptor [18] This receptor has even higher identity between Xenopus and human than the Y1 receptor, but is more divergent in parts of the large third intracellular loop. Most of the differences between the Xenopus and human Y1 receptors reside in the amino-terminal extracellular segment, transmembrane region IV and extracellular loop 2 (Fig. 1). The differences suggest that these parts of the receptor have no or only minor direct interactions with the ligands. In contrast, most other parts of the receptor are highly conserved, and the replacements that have occurred are usually of conservative nature even in the loops. This suggests that there is strong conservative selection pressure on these parts of the receptor, probably both for ligand-binding residues and residues that are involved in the conformation of the transmembrane regions and G-protein interactions. Recently, Walker and colleagues [19] identified by site-directed mutagenesis several aspartate residues in the extracellular loops of the human Y1 receptor that seem to be important for ligand binding. Complete loss of NPY binding was observed for the mutations D194A (D194 mutated to A), D200A, D205A and D287A. These mutations correspond to XY1 189, 195 and 200 in extracellular I
I
I
loop 3 and 283 in extracellular loop 4. It was hypothesized that at these positions salt bridges are formed with human NPY positions Arg-19, Arg-25, Arg-33, and Arg-35. Xenopus NPY differs from human NPY at position Arg-19, and has instead Lys (which is probably the ancestral amino acid; see Ref. [20]). This position was suggested to interact with Asp194 in the human Y1 receptor. In Xenopus this position (189) has Gly, a neutral amino acid. Thus, no salt bridge can be formed between these two amino acids in Xenopus. Possibly the absence of Asp at position 189 (in Xenopus) may be compensated for by either or both of two other negative residues in the same loop at positions 196 (Asp) and 199 (Glu). Additional studies are required to confirm the proposed ligand-receptor interactions, for instance by changing the other three Asp residues in the human Y1 receptor to Glu to maintain the negative charge and by replacing Xenopus Y1 positions 196 and 199. Furthermore, it is interesting to note that the less conserved amino terminus has two conserved acidic residues at positions Glu-10 (Xenopus pos. 8) and Asp-29 (Xenopus pos. 27). However, as neither of these positions affected binding when mutated to Ala [19] there clearly can be other reasons for maintaining extracellular charged residues than interaction with ligands. A conserved aromatic residue in the third intracellular loop is interesting with respect to the work by Bliiml and colleagues [21,22]. Their study on the rat muscarinic receptor M3 shows that a Tyr-residue (bold and underlined) in the sequence YWRIYKETEKRT, is important in G-protein coupling to the PI pathway. Site-directed mutagenesis has shown that the M3 receptor requires an aromatic residue, Tyr, Phe or Trp, in this position to get efficient PI coupling. It has been suggested that this is a general recognition motif for G-proteins [23,24] since various Gs, Gq and Gi-coupled receptors have this aromatic residue. Also the
I
I I f I 1 I I TM I TM I I MAN: MN~STLFSQVENHS-VHSNFSEKNAQLLAFEND~CHLpLAMIFTLALAY.GAVIILG~SGNLALIIIILKQKEMRNVTNILIVNLSFSDLLVAIMCL~FTFv RAT: ....7.R Y Y V N-SPF V ~ V MOU: ..;_ K I Y A N-SP V ~ V XEN: _f.._ Y - - L PN I GNITFPI--SE - A P L ~ AT LI
aa 99 98 98 94
TM I I I TM IV MAN: YT~MDHwVFGEAMCKLNPFVQCvSITvSIFSLvLIAVERHQLIINPRG~RPNNRHAYVGIAvIWvLAVASSLPFLIY~VMTDEPFQNvTLDAYKDKYvCF RAT: T I T V IL S A F MOU: T I T V IL S A F XEN: I V EYI V I CF T GF M C T LMM S L K IS S IG L
199 198 198 194
TM V TM V l MAN: DQFPSDSHRLSYTTLLLVLQYFGPLCFIFICYFKIYIRLKRRNNMMDKMRDNKYRSSETKRINIMLLSIvvAFAVC•LPLTIFNTvFD•NHQIIATCNHN RAT: K I S V MOU: K I S XEN: ED E KF FI L V T FL I G L FF EAV
299 298 298 294
TM V I I MAN : LLFLLCHLTAM•STC•NP•FYGFLNKNFQRDLQ•FFNFCDFRSRDDDYET•A•STMHT•V•KTSLKQASPVAFKKINNNDDNEK•* RAT: SM MOU: SM XEN: I E I *
* V*
384 382
382 366
Fig. 1. Alignment of NPY receptors from man, rat, mouse, and Xenopus. The human Y1 receptor shares 81% identity to the Xenopus Y1 receptor in the region spanning TMI-TMVII. The amino-terminal and the carboxy-terminal parts of these receptors share 47% and 96% identity, respectively. Dotted underlinings indicate putative glycosylation sites. * denotes termination.
441
A.G. Blomqvist et al. /Biochimica et Biophysica Acta 1261 (1995) 439-441
Xenopus Yl-receptor (YTKIFLRLKRRN) and the three mammalian Yl-receptors (YF-KIYIRLKRRN) have an aromatic residue in this position. Another conserved sequence of the Y1 receptor family is the ERH motif (ERItQLIIN) in the beginning of the second intracellular loop. In other peptide receptors this sequence is usually DRY or alternatively DRH, DRF, DRC, ERY, or ERW. Thus, the ERH motif seems to be unique for Yl-receptors. It will be interesting to see whether other NPY/PYY-receptors subtypes also have the ERH motif. The cloned Xenopus laevis Y1 receptor has been cloned in different expression vectors (pTEJ8, pcDNAI/Neo, pCD-PS) where the (GA)~s repeat in the 5'-untranslated region of the cDNA has been deleted, as this segment may compromise the expression efficiency. Nevertheless, the level of expression has been too low to give satisfying levels of expressed protein for binding studies. The human Y1 receptor has also been found to be difficult to express when transfected transiently in contrast to the rat Y1 receptor, that works sufficiently well. Sequence comparisons reveal that the sequence motif 'ATIq'A' is present in the Xenopus sequence five times, in the human receptor sequence four times, and in the mouse and rat receptor sequences only once. This motif has been shown to be a target for RNA-destabilizing enzymes and may therefore rapidly decrease the half-life of the mRNA [25]. An example of a cDNA with motifs for mRNA degradation within the coding regions is c-fos [25,26]. Another possibility is the presence of hairpin loops in the mRNA that decrease the translation efficiency. A third explanation might be that the cloned Xenopus Y1 receptor is non-functional, because Xenopus laevis has a tetraploid genome and numerous loci are present in duplicate. However, for another Xenopus receptors that exists in duplicate, both copies have been found to be functional [12]. Furthermore, it would seem unlikely that a 366 amino acids long sequence would not contain any frameshift- a n d / o r nonsense mutations if it does not have functional role. Subclones of the XYI-1 clone are currently used for in situ hybridization to study the cellular localization and level of expression of the Y1 receptor in the brain and pituitary of Xenopus laevis during background adaptation of the animal. This is of physiological significance because NPY is an important regulator of the adaptation process and it has recently been shown that the amounts of NPY peptide and mRNA in neurons of the suprachiasmatic nucleus differ greatly between black- and white-adapted animals [27]. We are grateful to Ann-Sofie EngstriSm, Charlotte Rouppe van der Voort and Maarten van Riel for their contributions to this work. This work has been supported by grants from the Swedish Natural Science Research Council (B-BU 8524-320), from the Netherlands Organization for Scientific Research (NWO), and from the Euro-
pean Community (BIOT-91-0302 BCHRXCT 920017).
and
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