Molecular Cloning of the cDNAs Encoding a Novel Insulin-Like Growth Factor-Binding Protein from Rat and Human

Abstract

cDNA clones encoding a novel insulin-like growth factor-binding protein (IGFBP) purified from rat serum and human bone cell-conditioned medium have been isolated from rat liver and human placenta, liver, and ovary cDNA libraries. The deduced amino acid sequences of the cDNAs revealed a mature polypeptide consisting of 233 amino acids for the rat, while the human structure contains an additional four-amino acid sequence in the middle region of the molecule. This protein, now proposed to be named IGFBP-4, contains two extra cysteines compared with the previously characterized IGFBP-1, -2, and -3, but the alignment of the remaining 18 cysteines is conserved across the four IGFBPs. Amino acid sequence comparison among the four binding proteins within the rat species demonstrated that both the amino- and carboxy-terminal one thirds of the molecules are highly conserved, while the middle one third region, where no cysteines are present except for the two that exist in IGFBP-4, is the most divergent. The overall sequence homology among the four rat IGFBPs is very similar (53-59%), suggesting that their individual genes diverged from a single ancestral gene at about the same evolutionary time point. Northern analysis of the IGFBP-4 mRNA in rat tissue demonstrated that transcription of the IGFBP-4 gene is highly active in the liver, although a single 2.6-kilobase IGFBP-4 mRNA band was detectable in all tissues examined, including adrenal, testis, spleen, heart, lung, kidney, liver, stomach, hypothalamus, and brain cortex.

Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1

“…6), including ovary from 1-year-old coho salmon (data not shown). Relative gene expression of 22 kDa IGFBP by RT-PCR was compared with the intensity of the plasma 22 kDa band on Western ligand Póvoa et al (1984), Shimasaki et al (1990), Maures and Duan (2002) and Bauchat et al (2001) respectively. Amino-acid residues identical to those of salmon 22 kDa IGFBP are shaded.…”

Targeted Expression of a Protease-resistant IGFBP-4 Mutant in Smooth Muscle of Transgenic Mice Results in IGFBP-4 Stabilization and Smooth Muscle Hypotrophy

“…Production of IGFBP-4 Mutant cDNA (IGFBP-4.7A)-A cDNA construct encoding a mutant IGFBP-4 in which all seven basic residues between Lys-120 and Lys-132 were exchanged for Ala residues was produced by stepwise mutagenesis of a cDNA construct originally provided by S. Shimasaki et al (30) (Fig. 1).…”

“…The extracts were run on 10% Laemmli gels under reducing conditions. The blots were blocked with a Tris-buffered saline solution containing 0.05% Tween 20 and nonfat dry milk and incubated with either 1:5000 rabbit anti-rat IGFBP4 antibody (516.F) generated against residues 81-100 of the rodent sequence (6,30) in the same blocking solution plus 0.01% thimerosal at room temperature for 16 h. IGFBP-4 was visualized by standard ECL techniques as described (33) and quantified by phosphorimager.…”

IGF-binding protein-4: biochemical characteristics and functional consequences

“…It is the second most abundant IGFBP in adult rat serum after IGFBP-3. Northern blot analysis revealed that IGFBP-4 mRNA is widely expressed in adult rat tissues, including adrenal gland, testis, spleen, heart, liver, lung, kidney, stomach, hypothalamus and brain cortex, with liver being the site of the highest expression (Shimasaki et al 1990). The expression of IGFBP-4 was also examined in rat small intestine (Shoubridge et al 2001), smooth muscle (Smith et al 2001), skeletal muscle ( Jennische & Hall 2000), pancreas (Hill et al 1999), uterus and placenta (Cerro & Pintar 1997), mouse spinal cord (Arnold et al 2000), mouse and human thymus (Li et al 1996), human prostate (Thomas et al 2000), bone (Mohan et al 1995a) and ovary (Zhou & Bondy 1993, el Roeiy et al 1994) of several species.…”