Growth hormone secretagogue related receptors and nucleic acids

Details Growth hormone secretagogue related receptors and nucleic acids Growth hormone secretagogue related receptors and nucleic acids

2001-03-15

2003-07-29

Mertz, Prema (Department: 1647)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S071100, C435S471000, C435S252300, C435S325000, C435S320100, C536S023500, C530S350000

Reexamination Certificate

active

06599718

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a new family of receptors, growth hormone secretagogue-related receptors (GHSR-Rs), nucleic acids encoding these receptors, and to the use of these receptors to identify ligands that modulate GHSR-R function.
BACKGROUND OF THE INVENTION
Growth hormone secretagogues (GHSs) and secretagogue-like compounds, both peptide and non-peptide, bind to and exert their biological effects (i.e., release of growth hormone (GH)) through a G protein-coupled receptor (GPC-R) distinct from the receptors for growth hormone releasing hormone (GHRH) and somatostatin (SST) (Pong et al., 1996
Mol. Endocrin.
10:57-61). The molecular cloning of the growth hormone secretagogue receptor (GHS-R) capitalized on the pivotal observation that GHSs transduce their signal through activation of the phospholipase C pathway (Cheng et al., 1991
Endocrinology
129:3337-3342; Howard et al., 1996
Science
273:974-977). cDNA and genomic DNA cloning from human, swine, and rat showed that the GHS-R is a remarkably conserved protein of 364/366 acids containing 7 putative alpha-helical transmembrane (TM) domains, a signature feature of GPC-R's (Howard et al. 1996; McKee et al., 1997
Mol. Endocrin.
11:415-423). In all species evaluated, the GHS-R is encoded by a single highly-conserved gene containing one intron, placed at the C-terminal end of TM domain 5. The GHS-R exhibits the highest sequence similarity to the receptors for neurotensin (NT-R) with sequence identity of 34%. The biology of the growth hormone secretagogues (GHSs) is still in a relatively early stage of development. Research is focused on identification of the GHS natural ligand system and understanding the role of the GHS-R in brain regions (substantia nigra, dentate gyrus, hippocampus) other than those traditionally thought to be involved in GH secretion (Bennett et al. 1997; Guan et al. 1997).
The identification of other G-protein coupled receptors points to the existence of a new natural ligand pathway perhaps divergent from the neuropeptide neurotensin and the GHS natural ligand. Two new human full-length GPC-R's entitled GPR38 and GPR39 were cloned having 52% and 32% protein sequence identity to the human GHS-R, respectively (McKee et al. 1997).
It would be desirable to identify other GPC-Rs perhaps impactful on GH release and elucidate their functions. It would also be desirable to identify ligands particular to these receptors that play an important role in these and other associated pathways.
One peptide for which receptors have not been molecularly identified to date is neuromedin U. Neuromedin U (NMU) is a neuropeptide first isolated from porcine spinal cord in two molecular forms, one containing 25 amino acids (NMU-25) and the other one 8 amino acids (NMU-8); Minamino et al., 1985
Biochem Biophs Res Commun
130:1078-85. It was subsequently isolated from rat (NMU-23), human (NMU-25), frog (NMU-25), dog (NMU-8 and NMU-25), rabbit (NMU-25), and chicken (NMU-25); Domin et al., 1986
Biochem Biophys Res Commun
140:1127-34; Conlon et al., 1988
J Neurochem
51:988-91; Minamino et al., 1988
Biochem Biophy Res Commun
156:355-60; Domin et al., 1989
J Biol Chem
26420881-5; O'Harte et al., 1991
Peptides
12:11-5; Kage et al., 1991
Regul Pept
33:191-8; and Domin et al., 1991
Regul Pept
41: 1-8. Mammalian NMUs share a common C-terminal sequence-Phe-Leu-Phe-Arg-Pro-Arg-Asn-amide which appears to be essential for its biological activities. NMU is distributed both in the gastrointestinal tract and the central nervous system (CNS).
In the rat, the highest concentration of NMU was found in the ileum, followed by the pituitary, hypothalamus, spinal cord, thyroid, and the genitourinary tract. Immunohistochemistry studies showed that NMU immunoreactivity in the gut was only found in nerve fibers, mainly in the myenteric and submucous plexuses, and in the mucosa of all areas except stomach while no NMU immunoreactivity was found in endocrine cells. In the rat brain, NMU immunoreactivity was found in fibers widespread throughout the brain with the exception of the cerebellum.
Human and rat genes encoding NMU precursor have been isolated. Both encode NMU at the C-terminus and other potential peptide products in the middle; Lo et al., 1992
Mol Endocrinol
6:1538-44; Austin et al., 1995
J Mol Endocrinol
14:157-69.
High affinity NMU binding was characterized in rat uterus, and was shown to be sensitive to GTP-&ggr;-S (Nandha et al., 1993
Endocrinology
133:482-6), suggesting the receptor for NMU was a G-protein coupled receptor. However, no receptor of NMU has been molecularly identified so far.
The physiological role of NMU remains largely unrecognized. It can cause potent contraction of smooth muscle, increase arterial blood pressure, modify intestinal ion transport, and at low doses stimulates the function and growth of the adrenal cortex. NMU was also shown to reduce the blood flow in superior enteric artery and portal vein while increase blood flow slightly in pancreatic tissue. Nevertheless, NMU is the only neuromedin without a receptor cloned nor a great deal of knowledge obtained concerning its pharmacology and physiology.
It would be most desirable to identify a G-protein coupled receptor responsive to neuromedin U or ligands sufficiently similar thereto. A receptor responsive to neuromedin U would greatly facilitate our understanding of the physiological mechanisms of neuromedin U and other ligands sufficiently similar thereto.
SUMMARY OF THE INVENTION
This invention relates to a new family of G protein-coupled receptors, growth hormone secretagogue-related receptors (GHSR-Rs) free from receptor-associated proteins, which exhibit moderate protein sequence identity (33 and 29%) to both the growth hormone secretagogue receptor (GHS-R) and the neurotensin receptor (NT-R) type 1, respectively. Particularly, the full-length mouse and human GHSR-Rs have been identified. These newly identified receptors are expressed in a diverse set of tissues. A further aspect of this invention is the above receptors which are isolated or purified.
Another aspect of this invention are GHSR-Rs which are encoded by substantially the same nucleic acid sequence, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native form. These variant forms may have different and/or additional functions in animal physiology or in vitro in cell based assays.
Growth hormone secretagogue related receptors (GHSR-Rs) are proteins containing various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify (e.g., by deletion) many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. This invention specifically includes such modified functionally equivalent GHSR-Rs as well as receptors comprising the binding domain of a GHSR-R of this invention.
Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.
Another aspect of this invention are nucleic acids which encode growth hormone secretagogue related receptors (GHSR-Rs). More specifically, the invention relates to nucleic acids comprising the sequences of SEQ ID NOs: 1 and 3 as well as those which hybridize to same under highly stringent conditions. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode a GHSR-R.
Yet another aspect of this invention

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