(Received for publication, September 15, 1995)
From the
In order to understand the molecular mechanisms that underlie the co-evolution of related yet functionally distinct peptide-receptor pairs, we study receptors for the vasopressin-related peptide Lys-conopressin in the mollusc Lymnaea stagnalis. In addition to a previously cloned Lys-conopressin receptor (LSCPR1), we have now identified a novel Lys-conopressin receptor subtype, named LSCPR2. The two receptors have a differential distribution in the reproductive organs and the brain, which suggests that they are involved in the control of distinct aspects of reproduction and mediate transmitter-like and/or modulatory effects of Lys-conopressin on different types of central neurons. In contrast to LSCPR1, LSCPR2 is maximally activated by both Lys-conopressin and Ile-conopressin, an oxytocin-like synthetic analog of Lys-conopressin. Together with a study of the phylogenetic relationships of Lys-conopressin receptors and their vertebrate counterparts, these data suggest that LSCPR2 represents an ancestral receptor to the vasopressin/oxytocin receptor family in the vertebrates. Based on our findings, we provide a theory of the molecular co-evolution of the functionally distinct ligand-receptor pairs of the vasopressin/oxytocin superfamily of bioactive peptides.
Peptide receptors form an important subclass of the otherwise
diverse superfamily of G protein-coupled ()receptors that
all have a seven-transmembrane segment topology in common (for review,
see (1) ). This receptor type forms a component of a modular
system for the transduction of extracellular signals across the cell
membrane and the subsequent conversion of these signals to an
intracellular second messenger pathway via the activation of
heterotrimeric G proteins (for review, see (2) ). Molecular
evolutionary mechanisms such as gene duplication and subsequent
mutation of the resulting genes have resulted in the formation of
families of related yet distinct peptides (3) and peptide
receptors (4) . In the nervous and endocrine systems, many
peptide isoforms bind to distinct receptor subtypes that mediate the
specific cellular actions that underlie a large variety of behavioral
and physiological processes. Peptide-receptor and receptor-effector
interactions are of critical importance for peptide function, and many
diseases are linked to malfunctions of these interactions(5) .
However, the great variety in structure of both peptides and receptors
has hampered the development of a coherent theory explaining the
molecular basis of co-evolution of specifically interacting
peptide-receptor pairs.
The vasopressin/oxytocin superfamily of
peptides and their cognate receptors offer an attractive model for the
study of specificity of peptide-receptor interactions and the
co-evolution of related peptide-receptor pairs(6) . These
peptides and receptors occur throughout the entire animal kingdom (for
review, see (7) ), allowing detailed comparison of their
structural features and experimental testing of putative specificity
determinants. The peptides of the superfamily are surprisingly alike,
sharing at least 5 out of 9 residues and a disulfide-linked ring
structure that put severe constraints on conformational
flexibility(8, 9) . In the vertebrates, gene
duplication gave rise to related yet distinct vasopressin and oxytocin
genes(10) . The different functions of vasopressin and oxytocin
are mediated by genetically distinct receptor
subtypes(11, 12) . The V2 vasopressin receptor
mediates the antidiuretic effects of vasopressin and couples positively
to adenylate
cyclase(13, 14, 15, 16) . By
contrast, the V1a (17, 18) and the V1b (19) vasopressin receptors, which mediate the effects of
vasopressin on liver glycogenolysis and on adrenocorticotropin release,
respectively, as well as the oxytocin
receptor(15, 20, 21) , which mediates the
various central and peripheral functions in reproduction of oxytocin,
all couple to the inositol trisphosphate/Ca signal
transduction pathway. The discriminative binding of vasopressin and
oxytocin to their respective receptors is dictated to a large extent by
the amino acid residue at position 8. At this position, the family of
vasopressin and related peptides have a basic residue, whereas oxytocin
and related peptides have a neutral, in most cases aliphatic
residue(7) .
In the invertebrates, on the other hand, only one gene is present that is considered the present-day representative of the ancestral peptide gene to the vasopressin/oxytocin superfamily(9) . In the mollusc Lymnaea stagnalis, this gene encodes Lys-conopressin, a peptide that is structurally related to vasopressin and that has a wide distribution throughout the invertebrate phyla(9, 22, 23, 24, 25) . In Lys-conopressin, the chemical nature of the amino acid residue at position 8 is not important for peptide function, since replacement of this basic residue (lysine) by an aliphatic one (isoleucine) does not affect the potency of binding of the peptide to its receptor(9) . These studies also show that Lys-conopressin, although structurally related to vasopressin, controls reproductive functions that are clearly analogous to the central and myoregulatory functions of oxytocin in vertebrate reproduction. In addition, Lys-conopressin serves vasopressin-like functions in the control of carbohydrate metabolism (26) . Together, these observations suggest that functionally distinct vasopressin and oxytocin receptors may have evolved from a nondiscriminative Lys-conopressin receptor-like receptor by gene duplication and subsequent introduction of specificity determinants that enable discriminative binding of vasopressin and oxytocin on the basis of the chemical nature of amino acid residue 8.
We have recently cloned a Lys-conopressin receptor from the Lymnaea vas deferens, named L. stagnalis conopressin receptor (LSCPR), that displays discriminative binding properties with respect to residue 8 in Lys-conopressin(26) . Here we report the cloning of a second Lys-conopressin receptor, named LSCPR2, that unlike the previously identified receptor (now renamed LSCPR1), does not discriminate between Lys-conopressin and its oxytocin-like synthetic analog Ile-conopressin. We show that vasopressin and oxytocin receptors may have evolved from an LSCPR2-like ancestral receptor, and propose a mechanism for the molecular evolution of specificity in the peptide-receptor pairs of the vasopressin/oxytocin superfamily. Finally, we discuss the role that preexisting receptor subtypes may have had in the historic development of functionally distinct vasopressin and oxytocin lineages.
Figure 1: The primary structure of LSCPR2. A, nucleotide sequence and deduced amino acid sequence of clone pBSCPR2. Only the open reading frame and a part of the 5`-untranslated leader sequence are shown. Nucleotides and amino acids are numbered at the right hand side, starting at the first ATG and the corresponding Met residue. Nucleotides upstream from the first ATG are indicated with negative numbers. An in-frame stop codon at position -45 is underlined. Putative transmembrane domains in the predicted protein sequence are shown in reversed contrast. Putative sites for N-linked glycosylation (Asn-X-Ser/Thr) are indicated by asterisks, and putative sites for phosphorylation by protein kinase C (Ser/Thr-X-Arg/Lys), casein kinase II (Ser/Thr-X-X-Asp/Glu) and cAMP-dependent protein kinase (Arg/Lys-Arg/Lys-X-Ser/Thr) are indicated by dots, triangles, and daggers, respectively. B, hydrophobicity plot of the LSCPR2 protein according to Kyte and Doolittle(36) . Numbers below the figure indicate amino acid residue positions in the LSCPR2 protein. The seven hydrophobic regions that represent putative transmembrane domains are shown in black.
An amino acid sequence alignment of LSCPR2, LSCPR1, and receptors for vasopressin, oxytocin, and vasotocin from vertebrates is presented in Fig. 2. In the region from the beginning of TM1 to the end of TM7, LSCPR2 has 43% sequence identity with the human oxytocin receptor, the rat V1a, and the human V1b vasopressin receptors and the fish vasotocin receptor, and 39% sequence identity with LSCPR1 and the rat V2 vasopressin receptor. Sequence identity is highest in the transmembrane domains, especially in TM7, whereas the N- and C-terminal domains as well as the third intracellular loop show hardly any sequence identity.
Figure 2: Alignment of the amino acid sequences of LSCPR2 and members of the vasopressin/oxytocin receptor family. The amino acid sequence of LSCPR2 is aligned with those of LSCPR1(26) , the human oxytocin receptor (HSOTR; (20) ), the rat V1a vasopressin receptor (RNAV1a; (17) ), the human V1b vasopressin receptor (HSAV1b; (19) ), the vasotocin receptor of the teleost fish Catostomus commersoni (CCVTR; (41) ), and the rat V2 vasopressin receptor (RNAV2; (14) ). Amino acid residues that are identical in all receptor proteins are shown in boldface. Asterisks indicate amino acid residues that are highly conserved in the vertebrate vasopressin/oxytocin receptor family, but not in other G protein-coupled receptors and have been suggested to be important in receptor-ligand interaction(6) . Bars indicate the seven putative transmembrane domains.
Several amino acid residues (indicated by asterisks in Fig. 2) that are conserved only among the members of the vasopressin/oxytocin receptor family are thought to be important in ligand binding(6) . In LSCPR2, these residues are either identical or conserved, with the exception of a threonine residue replacing the glycine residue in the sequence Pro-Trp-Gly (just before TM5). Interestingly, the two conserved proline residues in the sequences Gly-Pro-Asp and Pro-Trp-Gly (just before TM3 and TM5, respectively), which are replaced by aspartate residues in LSCPR1, are unchanged in LSCPR2.
Two adjacent cysteines that are found 15 residues downstream
of TM7 in the vasopressin and oxytocin receptors may be responsible for
anchoring the C terminus to the plasma membrane through palmitoylation,
as has been observed for the -adrenergic receptor(40) . In
LSCPR2, there are 4 adjacent cysteines present 4 residues further
downstream. These may be involved in linking the C terminus of the
receptor to the plasma membrane through a palmitoylation anchor.
Figure 3:
Functional expression of LSCPR2 in Xenopus oocytes. A, membrane current traces of an
oocyte expressing the LSCPR2 protein and being exposed to
10M vasopressin, oxytocin,
Arg-conopressin, Lys-conopressin, and Ile-conopressin (indicated by bars). Only application of conopressins elicited membrane
current responses, whereas vasopressin and oxytocin as well as all the
other vertebrate peptides tested were inactive. The initial fast inward
current was typically followed by an oscillatory current, which
represents intracellular calcium oscillations and lasted for about 5
min after washing out of the peptide. B, dose-response curves
of the effects of Lys-conopressin (black circles) and
Ile-conopressin (open squares) on the total membrane current.
The total membrane currents elicited by 1.5-min applications of either
of the peptides at different concentrations were integrated over time
and plotted semilogarithmically versus the peptide
concentration. The mean of three independent experiments is shown. The
EC
values are 86 nM for Lys-conopressin and 96
nM for Ile-conopressin.
Figure 4: Tissue distribution of LSCPR1 and LSCPR2 as determined by RT-PCR. LSCPR1 and LSCPR2 expression were analyzed in brain (lane 1), penis complex (lane 2), anterior vas deferens (lane 3), posterior vas deferens (lane 4), prostate gland (lane 5), spermoviduct (lane 6), ovotestis (lane 7), albumen gland (lane 8), oothecal gland (lane 9), muciparous gland (lane 10), kidney (lane 11), skin (lane 12), salivary gland (lane 13), and hepatopancreas (lane 14). The LSCPR1 transcript could be amplified from the brain and the anterior and posterior vas deferens, and the LSCPR2 transcript could be amplified from the brain, the spermoviduct and the posterior vas deferens. Lanes 15 and 16 contain PCR products derived from the LSCPR1 and LSCPR2 clones, respectively, and serve as positive controls for the PCR and the hybridization procedure.
Figure 5: Localization of LSCPR2 gene expression in the brain by in situ hybridization. A, section through the visceral ganglion; B, section through the right parietal ganglion showing two unidentified neurons, indicated by arrows, that express the LSCPR2 gene; C, section through the right cerebral ganglion ganglion showing a small group of unidentified neurons, indicated by arrows, that expresses the LSCPR2 gene. Neither of these neurons was found to express the LSCPR1 gene (not shown), nor could co-expression of LSCPR1 and LSCPR2 be detected in any other part of the brain. (Magnification, 250X.)
Figure 6: Phylogenetic relationships of the members of the vasopressin/oxytocin receptor family. Using an accepted mutation methodology (L. F. Kolakowski, Jr., and K. A. Rice, submitted for publication), the phylogenetic relationships of LSCPR1, LSCPR2, and related receptors from vertebrates were analyzed. The most parsimonious tree resulting from these analyses is shown. Numbers above the branches are the results of a bootstrap analysis and are confidence limits for the positions of the branches. The abbreviations used are as follows: V2R, V2 vasopressin receptor; V1aR, V1a vasopressin receptor; V1bR, V1b vasopressin receptor; VTR, vasotocin receptor; OTR, oxytocin receptor; ITR, isotocin receptor; CPR, conopressin receptor. The vasotocin receptors from the toad X. laevis and from the teleost fish Oncorhynchus kisutch, as well as the isotocin receptor from the teleost fish Astyanax fasciatus, have been cloned partially (41) and are classified as such on the basis of their positions in the tree. The Bovine rhodopsin sequence was utilized as an outgroup for the tree.
Compared with the vertebrate receptors, the LSCPR2
protein has the highest sequence identity with the mammalian V1a and
V1b vasopressin receptors(17, 19) , the mammalian
oxytocin receptor(20) , and the vasotocin receptor from teleost
fish(41) , which all couple to the inositol
trisphosphate/Ca signaling pathway. Sequence identity
with the mammalian V2 vasopressin receptor(13, 14) ,
which is coupled to adenylate cyclase, is less. Furthermore, Xenopus oocytes expressing the LSCPR2 protein respond to
application of Lys-conopressin by displaying a dose-dependent inward
chloride current (Fig. 3), a response that is characteristic for
receptors that activate the inositol trisphosphate/Ca
signal transduction pathway(30) . These findings indicate
that most likely LSCPR2 is coupled to a G protein that activates the
inositol trisphosphate/Ca
signal transduction
pathway. LSCPR1 has similar signal transduction characteristics,
however, half-maximal receptor activation is reached at four times
lower concentrations of Lys-conopressin(26) . The first and
second extracellular loops of the vasopressin and oxytocin receptors
have been suggested to form a ligand binding pocket(6) .
Interestingly, the corresponding loops in LSCPR1 contain two unique
aspartate residues that are absent in LSCPR2 (Fig. 2). Since
conopressins differ from other vasopressin- and oxytocin-related
peptides in that they have a negatively charged arginine residue at
position 4(22) , we have suggested that either of these
positively charged aspartate residues may be important in the
interaction of LSCPR1 with this arginine residue(26) . The
absence of the aspartate residues in LSCPR2 might explain the fact that
this receptor is less sensitive to Lys-conopressin than LSCPR1.
The differential distribution of the two Lys-conopressin receptors suggests that they must have different functions. In the periphery, they are most likely involved in the regulation of distinct aspects of reproduction, whereas in the brain, they probably mediate transmitter-like or modulatory effects of Lys-conopressin on different types of neurons. A possible explanation for the existence of multiple conopressin receptors is that it allows a stimulus-dependent differential expression of the receptor genes in various target tissues and cells, thus providing a mechanism for a spatio-temporal co-ordination of Lys-conopressin actions in otherwise conflicting types of behavior and physiological processes. Similarly, transcriptional regulation of the human oxytocin receptor gene plays an important role in the physiologically relevant increase in the number of oxytocin receptors in the uterus of pregnant females at the onset of labor(20) . In addition, the difference in the sensitivity of the two receptors to Lys-conopressin that we observed may be physiologically relevant in this respect as well.
As we have shown, specificity determinants in the small and conformationally constrainted peptides of the vasopressin/oxytocin superfamily evolved in a different way. Residues 1-6 of these peptides are important for high affinity binding to the receptor(26) , whereas specific receptor activation depends on the chemical nature of the single amino acid residue at position 8. In contrast to the development of luteinizing hormone and follicle-stimulating hormone and their receptors, where separation of large specificity domains in both the peptides and the receptors allowed for a continuous refinement of specificity during evolution(45) , functionally distinct vasopressin and oxytocin peptides can only have evolved by trial-and-error mutation of residue 8 in the presence of a nondiscriminative receptor such as LSCPR2. This trial-and-error phase may be reflected in various present day species of cartilaginous fish. Unlike the evolutionary stable vasopressin lineage, oxytocin-like peptides are very diverse in this primitive group of the vertebrates(7) . To none of these peptides has a function been assigned, and they have very poor uterotonic activity in mammals(46) . Hence, it will be of interest to examine whether functional receptors exist for these peptides. If not, they must be considered relicts of an early step in the historic development of the oxytocin lineage of bioactive peptides.
To further demonstrate that
a nondiscriminative LSCPR2-like receptor may have been ancestral to the
vasopressin/oxytocin receptor family, we studied the phylogenetic
relationships of the Lys-conopressin receptors and their vertebrate
counterparts using parsimony analysis. Phylogenetic reconstruction is
nowadays accepted as a valid method to study the evolutionary histories
of families of related receptors and provides useful insights into the
structures and functions of the individual members(47) . ()Our data indicate that LSCPR2 is indeed more closely
related to the vertebrate vasopressin and oxytocin receptors than
LSCPR1 (Fig. 6). Therefore, LSCPR2 most likely is a present day
representative of the ancestral receptor to the vertebrate receptors.
In addition, the phylogenetic analysis shows that LSCPR1 and LSCPR2
probably result from an ancient receptor duplication that occurred
before separate receptor types in the vertebrates evolved. Thus,
multiple receptors may have existed before separate lineages of
vasopressin- and oxytocin-related peptides evolved. Since the evolution
of functionally distinct peptide lineages from a common ligand-receptor
pair requires not only the introduction of specificity determinants on
both the peptides and the receptors but also a differential cellular
pattern of expression of distinct receptor types, we suggest that
preexistence of differentially expressed receptor subtypes was of
significant importance in the functional divergence of vasopressin and
oxytocin in the vertebrates.
As in the peptides of the vasopressin/oxytocin superfamily, specificity determinants in the corresponding receptors are probably restricted to small parts of the receptor molecules(6) . LSCPR2 can be very useful in the search for these determinants. Experiments involving chimeric receptors and swapping of putative binding domains might mimmick the evolutionary process of introducing specificity determinants in a nondiscriminative ancestral receptor such as LSCPR2, and increase our understanding of the molecular basis of peptide-receptor interactions. This then might enable the rational design of highly potent and specific receptor agonists and antagonists.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40491[GenBank].