TIP39 A Potent and Selective ligand
of PTH2 Receptor
TIP 7-39, a selective antagonist for PTH1-R
TIP39 (tubero-infundibular peptide of 39 residues), a new neuropeptide
isolated from bovine brain, was identified as a potent and selective
ligand for PTH2 receptor.
TIP39 potently activates the human and rat PTH2 receptors
but has little or no effect at PTH1 receptor by TIP39
results in a twofold greater accumulation in cAMP than that
elicited by PTH, and TIP39 is 100-fold more potent than PTH.
By contrast, TIP39 and PTH have similar potency and efficacy
at the human PTH2 receptor. Because TIP39 was purified
from brain and PTH2 receptor expression id highest
in the brain, it seems likely that , at least in the CNS, a
homolog of bovine TIP39 acts on the human PTH2 receptor.
TIP39 might be the physiologically relevant PTH2
receptor ligand and be involved in the modulation of processes
that range from pituitary hormone release to nociception and
regulation of blood pressure. Usdin T. B. TiPS 21, 128-130 (April,
2000)
Rhodamine-TIP39 and FAM-TIP39 bind to human PTH-2R
Ted
B. Usdin, M.D., Ph.D. Laboratory of Genetics, NIMH
The release of CRF from medial basal hypothalamic explants
by TIP39 1 nM, 10 nM,
and 100 nM; PTH 100 nM
and 56 mM potassium as a percentage
of basal release. Basal, peptide then potassium 56 mM
incubation periods each of 45 min duration. Values are shown
as mean ± SEM; *, P < 0.05
and ***, P < 0.01 by t test. H. L. Ward,
et al. Endocrinology Vol. 142, No. 8 3451-3456 (2001)
The release of plasma pituitary hormones 10 min after icv
injection of saline (open bars) TIP39 (closed bars)
1, 3, or 10 nmol and PTH (1?4) 10 nmol (striped
bars). A, ACTH values are shown as mean pg/ml ± SEM;
B, LH values are shown as mean ng/ml ± SEM.
*, P < 0.05 and **, P < 0.01 by ANOVA
with post hoc Bonferroni correction. H. L. Ward,
et al. Endocrinology Vol. 142, No. 8 3451-3456 (2001)
A, Amino acid sequence alignment for the human
and murine TIP39 precursors. Residues that are identical (dark
shade) or similar (light shade) in human and mouse
TIP39 are shown; the black bar depicts the secreted
peptide with the first residue denoted as "+1". B, Kyte/Doolittle
hydrophobicity plot of the deduced human TIP39 precursor (upper
panel) and mouse TIP39 precursor (lower panel)
amino acid sequence. The thick black bar depicts the
secreted peptide; the position of the first residue is denoted
as "+1". The ordinate indicates relative hydrophobicity, with
more positive values corresponding to increased hydrophobicity.
Markus R. et al. Endocrinology
Vol. 143, No. 3 1047-1057 (2002).
Comparison of the gene structure for human TIP39, human PTH,
and human PTHrP. Boxed areas are exons and their names
are shown underneath (because the start of exon U1 of the
TIP39 gene is unknown, the box is open on the left side),
white boxes denote presequences, black boxes
denote prosequences (for TIP39 presumed), gray stippled
boxes denote the mature sequences; noncoding regions are
shown as striped boxes. The small striped boxes
preceding the white boxes denote untranslated exonic
sequences (4 bp for TIP39; 5 bp for PTH; 22 bp for PTHrP).
The positions of the initiator methionine based on the secreted
peptide are noted above the graphs; the positions where prosequences
are interrupted by an intron are noted above the graph.
+1 denotes the relative position of the beginning of the secreted
peptide. Markus
R. et al. Endocrinology
Vol. 143, No. 3 1047-1057 (2002).
A, Ligand-stimulated cAMP accumulation in hPR2–20 LLCPK1
cells stably expressing the recombinant human PTH2 receptor.
Cells were stimulated with increasing concentrations of human
TIP-(1–39) ()
or mouse TIP-(1–39) ().
Data are expressed as picomoles per well and represent the
results (mean ± SEM) of two independent
experiments; basal cAMP accumulation was 0.23 pmol/well. B,
Inhibition of agonist stimulated cAMP accumulation in hPR2–20
LLCPK1 cells. Cells were stimulated with approximately
half-maximal concentrations of human TIP-(1–39) ()
or PTH-(1?4) ()
in the absence or presence of increasing concentrations of
TIP-(9–39). Data are expressed as percentage of half-maximal
cAMP accumulation and represent the results (mean ± SEM)
of two independent experiments. Markus R. et al. Endocrinology
Vol. 143, No. 3 1047-1057 (2002).
Phylogenetic analysis indicating the evolutionary relationship
among precursor proteins of the TIP39, PTH, PTHrP, and secretin
families of peptides. A Neighbor-Joining phylogenetic analysis
using distance as the criteria is shown above (tree length
= 740, consistency index excluding uninformative residues
= 0.876, with 165 parsimony-informative characters). The bootstrap/jackknife
values from 10,000 replicates indicate support of a given
node where 95% is considered to be significant (36 37 ). A
Maximum Parsimony analysis using parsimony as the criteria
generated a similar phylogenetic relationship between PTH,
PTHrP, TIP39, secretin, and GIP (tree length = 746, consistency
index excluding uninformative residues = 0.846, with 165 parsimony-informative
characters). In addition to the Neighbor-Joining and Maximum
Parsimony phylogenetic analyses, Quartet puzzling using Maximum
Parsimony criteria and Star-decomposition (tree length = 739,
consistency index excluding uninformative characters = 0.855,
with 165 parsimony-informative characters) (35 36 ) support
the hypothesis that PTH and PTHrP are sister groups, and that
TIP39 is the sister group to this clade. PTH, PTHrP, and TIP39
thus form a superfamily, whereas secretin and VIP (not shown)
appears to be a sister group to this larger superfamily (accession
nos.: PTH (cat, AF309967; chick, M36522; cow, J00024; dog,
U15662; horse, AF134233; human, NM_000315; macaque, AF130257;
mouse, NM_020623; pig, X05722; and rat, NM_017044); PTHrP
(chick, X52131; dog, U15593; cow, P58073; human, J03580; mouse,
M60056; rabbit, AF219973; rat, NM_012636; sheep, AF327654;
fugu, AJ249391; sparus, AF197904); VIP (chick, U09350; mouse
AK018599; human XM_004381); secretin [mouse, X73580; pig,
M31496; human, XM_012014; and human GIP (NM_004123)]. Markus
R. et al. Endocrinology
Vol. 143, No. 3 1047-1057 (2002).
A, Schematic representation of the known portions of the human
(upper panel) and the mouse (lower panel) TIP39
gene. The names of the different exons are indicated; the
sizes of exons (normal letters) and introns (italic
letters) are given in base pairs; the approximate positions
of the different PCR primers are shown (see Materials and
Methods); note that the positions of the universal AP1
and AP2 primers that were used for 5' RACE are arbitrary.
B, Splice donor/acceptor sites in the human and mouse gene
are shown; exonic nucleotides are shown in capital letters;
intronic nucleotides in lowercase letters; splice site
consensus nucleotides are in bold; the initiator ATG
in exon 1 is underlined. Markus R. et al. Endocrinology
Vol. 143, No. 3 1047-1057 (2002).
Hypothalamic releasing factors released from medial
basal hypothalamic explants
Hypothalamic releasing
factor
Basal period
TIP39 100 nM
56 mM
potassium
CRF
(pmol/explant)
2.9
± 0.4
4.4
± 0.61
4.6
± 0.61
AVP
(fmol/explant)
454.3
± 40.0
563.5
± 55.52
774.1
± 62.21
VIP
(fmol/explant)
73.4
± 9.6
85.6
± 8.43
101.4
± 13.11
LHRH
(fmol/explant)
19.3
± 2.5
28.5
± 5.13
39.5
± 4.22
TRH
(fmol/explant)
50.0
± 6.2
57.1
± 10.9
462.0
± 73.61
SST
(fmol/explant)
111.0
± 11.0
109.1
± 10.7
223.3
± 19.21
GRF
(pg/explant)
657.8
± 77.7
770.9
± 90.72
832.8
± 87.41
Basal, TIP39 100 nM
followed by potassium 56 mM incubation
periods each of 45 min duration. Values are shown as mean
± SEM. 1P < 0.001,
2P < 0.01, 3P <
0.05, by t test. H. L. Ward,
et al. Endocrinology Vol. 142, No. 8 3451-3456 (2001)
)
or mouse TIP-(1–39) (
).
Data are expressed as picomoles per well and represent the
results (mean ± SEM) of two independent
experiments; basal cAMP accumulation was 0.23 pmol/well. B,
Inhibition of agonist stimulated cAMP accumulation in hPR2–20
LLCPK1 cells. Cells were stimulated with approximately
half-maximal concentrations of human TIP-(1–39) (
)
in the absence or presence of increasing concentrations of
TIP-(9–39). Data are expressed as percentage of half-maximal
cAMP accumulation and represent the results (mean ± SEM)
of two independent experiments. 
