WO2000002918A1

WO2000002918A1 - Mouse growth hormone secretagogue receptor

Info

Publication number: WO2000002918A1
Application number: PCT/US1999/015375
Authority: WO
Inventors: Roy G. Smith; Leonardus H. T. Van Der Ploeg; Andrew D. Howard; Hui Zheng; Karen Kulju Mckee; Minghao Jiang
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1998-07-10
Filing date: 1999-07-08
Publication date: 2000-01-20
Anticipated expiration: 2001-01-10
Also published as: CA2335272A1; EP1097169A1; JP2002520337A

Abstract

A mouse growth hormone secretagogue receptor has been isolated, cloned and sequenced. This receptor is characteristic of the G-protein family of receptors. Mouse growth hormone secretagogue receptors may be used to screen and identify compounds which bind to the mouse growth hormone secretagogue receptor. Such compounds may be used in the treatment of conditions which occur when there is a shortage of growth hormone, such as observed in growth hormone deficient children, elderly patients with musculoskeletal impairment and those recovering from hip fracture and osteoporosis. Targeted disruption of the mouse GHS-R gene may prove useful in elucidation of the mechanism of action and role of the growth hormone secretagogues in human and animal physiology.

Description

TITLE OF THE INVENTION

MOUSE GROWTH HORMONE SECRETAGOGUE RECEPTOR

FIELD OF THE INVENTION This invention relates to a newly identified receptor, the mouse growth hormone secretagogue receptor (mGHS-R), nucleic acids encoding this receptor; and to the use of a mGHS-R to identify growth hormone secretagogues and compounds that modulate mGHS-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 this 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 protein of 364/366 amino acids containing 7 putative alpha-helical transmembrane (TM) domains, a signature feature of GPC-Rs (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 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).

It would be desirable to know the molecular structure of growth hormone secretagogue receptors in order to analyze this new receptor family and understand its normal physiological role in concert with the actions of GHRH and somatostatin. This could lead to a better understanding of the in vivo processes which occur upon ligand-receptor binding. Further, it would be desirable to use cloned-growth hormone secretagogue receptors as essential components of an assay system which can identify new growth hormone secretagogues which would confer a significant benefit on children and adults deficient in growth hormone, the frail elderly, those in post-hip fracture rehabilitation and post-operative recovery patients.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a novel receptor, mouse growth hormone secretagogue receptor (mGHS-R), which is free from receptor associated proteins. A further aspect of this invention is mGHS-R which is isolated or purified.

Another aspect of this invention is mGHS-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 receptors 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 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. Thus, this invention specifically includes modified functionally equivalent mGHS-Rs which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent mGHS-Rs which contain modified and/or deletions in other domains, which are not accompanied by a loss of functional activity.

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.

A further aspect of this invention are nucleic acids which encode a mouse growth hormone secretagogue receptor or a functional equivalent. 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 mGHS-R or a functional equivalent.

Yet another aspect of this invention relates to vectors which comprise nucleic acids encoding mGHS-R or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes, transposable elements and other forms of episomal or integrated DNA that can encode a mGHS-R. It is well within the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.

A further aspect of this invention are host cells which are transformed with a vector comprising a gene which encodes a mouse growth hormone secretagogue receptor or a functional equivalent. The host cell may or may not naturally express a GHS-R on the cell membrane. Preferably, once transformed, the host cells are able to express the mouse growth hormone secretagogue receptor or a functional equivalent on the cell membrane. Depending on the host cell, it may be desirable to adapt the DNA so that particular codons are used in order to optimize expression. Such adaptations are known in the art, and these nucleic acids are also included within the scope of this invention. Generally, mammalian cell lines, such as COS, HEK-293, CHO, HeLa, NS/0, CV-1, GC, GH3 or VERO cells are preferred host cells, but other cells and cell lines such as Xenopus oocytes or insect cells, may also be used.

Another aspect of this invention is a process for identifying nucleic acids encoding mouse growth hormone secretagogue related receptors comprising hybridizing a first nucleic acid encoding a mouse growth hormone secretagogue receptor with a second nucleic acid suspected of comprising nucleic acids encoding a growth hormone secretagogue receptor, wherein the hybridizing takes place under relaxed or moderate post hybridizational washing conditions; and identify areas of the second nucleic acid where hybridization occurred.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 is the DNA sequence encoding the mouse GHS-R, 5' and 3' flanking regions and the intron; SEQ ID NO:l.

FIGURE 2 is the DNA sequence encoding the open reading frame (ORF) of the mouse GHS-R; SEQ ID NO:2. FIGURE 3 is the deduced amino acid sequence of the mouse GHS- R; SEQ K> NO:3.

FIGURE 4 is an amino acid alignment of the mouse GHS-R with other GHS-R's from several species (human - SEQ ID NO:4, rat - SEQ ID NO: 5, and swine - SEQ ID NO:6).

As used throughout the specification and claims, the following definitions shall apply:

Growth Hormone Secretagogue - any compound or agent that directly or indirectly stimulates or increases the release of growth hormone in an animal.

Ligands— any molecule which binds to the mGHS-R of this invention. These ligands can have either agonist, partial agonist, partial antagonist or antagonist activity. Free from receptor-associated proteins— the receptor protein is not in a mixture or solution with other membrane receptor proteins.

Free from associated nucleic acids— the nucleic acid is not covalently linked to DNA which it is naturally covalently linked in the organism's chromosome. Isolated receptor— the protein is not in a mixture or solution with any other proteins.

Isolated nucleic acid— the nucleic acid is not in a mixture or solution with any other nucleic acid.

Functional equivalent— a receptor which does not have the exact same amino acid sequence of a naturally occurring mouse growth hormone secretagogue receptor due to alternative splicing, deletions, mutations, or additions, but retains at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor. Such derivatives will have a significant homology with a natural mGHS-R and can be detected by reduced stringency hybridization with a DNA sequence obtained from a mGHS-R. The nucleic acid encoding a functional equivalent has at least about 50% homology at the nucleotide level to a naturally occurring receptor nucleic acid.

Purified receptor— the receptor is at least about 95% pure.

Purified nucleic acid— the nucleic acid is at least about 95% pure. Standard or high stringency post hybridizational washing conditions - 6 X SSC at 55°C.

Moderate post hybridizational washing conditions —6 X SSC at 45°C. Relaxed post hybridizational washing conditions — 6 X SSC at

30°C.

The mouse isoform of the previously identified GHS-R was cloned from two genomic DNA libraries for the generation of a GHS-R knock-out mouse. This isoform has been shown to be functionally activated by secretagogues such as growth hormone releasing peptide GHRP-6 and MK-0677 through expression studies of the complete and contiguous open reading frame of mGHS-R using the aequorin biolumenescence assay. The proteins of this invention were found to have structural features which are typical of the 7-transmembrane domain (TM)- containing G-protein linked receptor superfamily (GPC-R' s or 7-TM receptors), including seven transmembrane regions, three intra- and extracellular loops, and the GPC-R protein signature sequence. Thus, mGHS-R, as an additional member of the growth hormone secretagogue family of receptors, constitutes a new member of the GPC-R family of receptors. Note not all regions are required for functioning, and therefore this invention also comprises functional receptors which lack one or more non-essential domains.

Sequence analysis of the mGHS-R revealed, further, the presence of a non-coding, intronic sequence at nt 790 corresponding to a splice-donor site (G/GT) (Fig. 1). This sequence insertion occurs two amino acids after the completion of the predicted transmembrane domain (TM) 5 (leucine-263), thus dividing the ORF of the mouse GHS-R into an amino-terminal segment (encompassing the extracellular domain, TM-1 through TM-5, and the first two intra-and extra-cellular loops) and a carboxyl-terminal segment containing TM-6, TM-7, the third intra- and extra-cellular loops, and the intracellular domain. The point of insertion and flanking DNA sequence are highly conserved between human, swine, rat and mouse. Comparison of the complete ORF encoding the murine GHS-R type la protein sequence (Fig. 4) to rat, human and swine GHS-R homologs reveals a high degree of sequence identity (mouse vs. rat, 99.5%; mouse vs. human 95%; mouse vs. swine 94%). The mGHS-Rs of this invention also share some sequence homology with previously cloned GPC-receptors including the rat and human neurotensin receptor (approximately 32 % identity) and the rat and human thyrotropin releasing hormone (TRH) receptor (approximately 29 % identity). The mGHS-R and fragments are immunogenic. Thus, another aspect of this invention is antibodies and antibody fragments which can bind to mGHS-R or a mGHS-R fragment. These antibodies may be monoclonal antibodies and produced using either hybridoma technology or recombinant methods. They may be used as part of assay systems or to deduce the function of a mGHS-R present on a cell membrane.

A further aspect of this invention are antisense oligonucleotides - nucleotides which can bind to mGHS-R nucleotides and modulate receptor function or expression.

A further aspect of this invention is a method of increasing the amount of mGHS-Rs on a cell membrane comprising, introducing into the cell a nucleic acid encoding a mGHS-R, and allowing expression of the mGHS-R.

A mGHS receptor, preferably immobilized on a solid support, may be used diagnostically for the determination of the concentration of growth hormone secretagogues, or metabolites thereof, in physiological fluids, e.g. body fluids, including serum, and tissue extracts, as for example in patients who are undergoing therapy with a growth hormone secretagogue.

The administration of a mGHS receptor to a patient may also be employed for purposes of amplifying the net effect of a growth hormone secretagogue by providing increased downstream signaling following administration of the growth hormone secretagogue thereby diminishing the required dosage of growth hormone secretagogue; or diminishing the effect of an overdosage of a growth hormone secretagogue during therapy.

Yet a further aspect of the present invention is a method of identifying ligands comprising contacting the mGHS-R with a compound suspected of being a ligand specific for said receptor and determining whether binding occurs, binding constituting a positive indication of the presence of a ligand.

Ligands detected using assays described herein may be used in the treatment of conditions which occur when there is a shortage of growth hormone, such as observed in growth hormone deficient children, elderly patients with musculo skeletal impairment and those recovering from hip fracture, and osteoporosis.

Targeted disruption of the mouse GHS-R gene may also prove useful in elucidation of the mechanism of action and role of the growth hormone secretagogues in human and animal physiology.

The following, non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1

Isolation of mouse GHS-R

A mouse (strain 129, liver) genomic library constructed in the vector lamda Fix II (Stratagene) was screened under moderate stringency hybridization conditions with a complete ORF probe derived from the swine GHS-R. Nylon filters repesenting 1.2 x 106 PFU were hybridized overnight at 58°C in 6 X SSC containing 10% dextran sulfate, 2% SDS, 0.5 M NaCl, and 100 μg/ml salmon sperm DNA with the random prime 32p_l_abeled swine GHS-R probe. Filters were washed in 4 X SSC, 1% SDS at room temerature for 20 minutes, 4 X SSC, 1% SDS at 55°C for 30 min, 2 X SSC, 1% SDS at 55°C for 30 min, and 2 X SSC, 1% SDS at 62°C for 30 min. Three positive clones were identified, phage DNA was isolated, and partial DNA sequencing performed to verify that they encoded the murine GHS-R gene. In addition, a mouse genomic library constructed in a B AC vector and gridded in a filter array (Genome Systems, Inc) was screened under moderate stringency hybridization conditions as given above with a complete ORF probe dervived from the human GHS- R. A positive clone was identified from the B AC library.

EXAMPLE 2

Sequencing Of Mouse GHS-R The BAC clone was sequenced with ABI Prism BigDye terminator cycle sequencing ready reaction mix (P/N 4303149; PE Applied Biosystems, Foster City, CA) using lμg DNA/reaction, 5% DMSO, 100 ng primer - standard cycle sequencing. Reactions were run on an ABI Prism 377 DNA Sequencer with XL Upgrade (ABI Prism 377XL). DNA from the positive lambda clones was prepared from a liquid lysate of the E. coli strain XLBlue MRA minus. For DNA sequencing, 500 ng of DNA was used under the same conditions as given above.

EXAMPLE 3

Analysis Of Mouse GHS-R Sequence

Sequence analysis revealed the presence of a non-coding, intronic sequence at nt 790 corresponding to a splice-donor site (G/GT) (Fig. 1). This sequence insertion occurs two amino acids after the completion of the predicted transmembrane domain (TM) 5 (leucine-263), thus dividing the ORF of the mouse

GHS-R into an amino-terminal segment (encompassing the extracellular domain, TM-1 through TM-5, and the first two intra-and extra-cellular loops) and a carboxyl-terminal segment containing TM-6, TM-7, the third intra- and extra-cellular loops, and the intracellular domain. The point of insertion and flanking DNA sequence are highly conserved between human, swine, rat and mouse. Comparison of the complete ORF encoding the murine GHS-R type la protein sequence (Fig. 4) to rat, human and swine GHS-R homologs reveals a high degree of sequence identity (mouse vs. rat, 99.5%; mouse vs. human 95%; mouse vs. swine 94%).

EXAMPLE 4

Construction Of Mouse GHS-R Expression Plasmid For expression studies in mammalian cells, a contiguous ORF

(Figs. 2 and 3) was assembled in the vector pcDNA-3 (Invitrogen) by overlapping PCR to remove the single intron present following nucleotide 790 of the ORF. To subclone, the Advantage HF PCR kit (K 1909-1; Clonetech Laboratories, Inc, Palo Alto, CA) was used under the following conditions: 94°C for lmin;, then 25 cycles of the following: 94°C for 15 sec, 55°C for 15 sec, and 68°C for 3 min. The primers used were: primer 1- 5'GGG CCC GAA TTC GCC GCC ATG TGG AAC GCG ACG CCC AGC 3' (SEQ ID NO: 7, including EcoR I site, Kozak initation sequence, and translational start Met); primer 2- 5'G4C CAC CAC AG C AAG CAT CTT CAC TGT CTG3' (SEQ ID NO:8; nucleotides shown in italic type overlap exon 2); primer 3- 5'AAG ATG CTTG CT GTG GTG GTG TTT GCT TTC ATC3' (SEQ ID NO:9; nucleotides shown in italic type overlap exon 1); and primer 4- 5'AGT TTA GCG GCC GCT CAT GTA TTG ATG CTC GAC TTT GT3' (SEQ ID NO: 10, including Not I site and stop codon). "Overlapping" PCR was performed. The first PCR reactions were performed with primers 1 and 2 (exon 1) or 3 and 4 (exon 2). The second PCR reactions were performed with primers 1 and 4 (ORF). The second product was digested with EcoRI and Notl, agarose gel purified, ethanol precipitated, phenol extracted, and ligated into pcDNA3 with Ready-to-Go T4 Ligase (27-0361-01; Pharmacia, Piscataway, NJ), and transformed into SCSI cells (200231; Stratagene, La Jolla, CA). DNA was isolated with Wizard Plus miniprep (A1460; Promega, Madison, WI) and 500ng was sequenced as above, but without DMSO.

EXAMPLE 5

Functional Activity Of Mouse GHS-R Measurement of mouse GHS-R expression in the aequorin- expressing stable reporter cell line 293-AEQ17 (Button et al., 1993 Cell Calcium 14:663-671.) was performed using a Luminoskan RT luminometer (Labsystems Inc., Gaithersburg, MD. 293-AEQ17 cells (8 x 105 cells plated 18 hr. before transfection in a T75 flask) were transfected with 22 μg of pcDNA-3 /mouse GHS-R plasmid DNA and 264 μg lipofectamine (Life Technologies). Forty hours after transfection, the apo- aequorin in the cells was charged for 1 hour with coelenterazine CP (10 μM) under reducing conditions (300 mM reduced glutathione) in ECB buffer (140 mM NaCl, 20 mM KC1, 20 mM HEPES-NaOH, pH=7.4, 5 mM glucose, 1 mM MgCl2, 1 mM CaCl2, 0.1 mg/ml bovine serum albumin). The cells were harvested, washed once in ECB medium and resuspended to 500,000 cells/ml. One hundred (100) μl of cell suspension (corresponding to 5x104 cells) was then injected into each well of a 96- well microtiter test plate, and the integrated light emission was recorded over 30 seconds, in 0.5 second units. Twenty (20) μl of lysis buffer (0.1% final Triton X-100 concentration) was then injected and the integrated light emission recorded over 10 seconds, in 0.5 second units. The "fractional response" values for each well were calculated by taking the ratio of the integrated response to the initial challenge to the total integrated luminescence including the Triton X-100 lysis response. Data were analyzed using GraphPad Prism software V.2.0 (GraphPad Software, San Diego, CA).

Claims

WHAT IS CLAIMED IS:

1. Mouse growth hormone secretagogue receptor, free from receptor-associated proteins.

2. Isolated mouse growth hormone secretagogue receptor.

3. A receptor according to Claim 1 or 2 which comprises SEQ ID NO:3.

4. A nucleic acid which encodes mouse growth hormone secretagogue receptor, said nucleic acid being free from associated nucleic acids.

5. A nucleic acid according to Claim 4 which is DNA.

6. A nucleic acid according to Claim 4 which comprises SEQ ID NO:l.

7. A nucleic acid according to Claim 4 which is RNA.

8. A vector comprising a nucleic acid which encodes a mouse growth hormone secretagogue receptor.

9. A vector according to Claim 8 which is selected from the group consisting of: plasmids, modified viruses, yeast artificial chromosomes, bacteriophages, cosmids and transposable elements.

10. A host cell comprising a vector according to Claim 8.

11. A method of identifying ligands which comprises:

(a) contacting the receptor of claim 3 with compounds suspected of being ligands specific for said receptor; and

(b) determining whether binding occurs, binding constituting a positive indication of the presence of a ligand.