Chimeric analogs derived from pairs of homologous proteins routinely exhibit activities found in one or both parents. We describe chimeras of two glycoprotein hormones, human chorionic gonadotropin (hCG) and human follitropin (hFSH), that exhibit activity unique to a third family member, human thyrotropin (hTSH). The results show that biological activity can be separated from hormone-specific amino acid residues. This is consistent with a model for the evolution of homologous ligand-receptor pairs involving gene duplication and the creation of inhibitory determinants that restrict binding. Disruption of these determinants can unmask activities characteristic of other members of a protein family. Combining portions of two ligands to create analogs with properties of a third family member can facilitate identifying key determinants of protein-protein interaction and may be a useful strategy for creating novel therapeutics. In the case of the glycoprotein hormones, this showed that two different hormone regions (i.e., the seat-belt and the intersubunit groove) appear to limit inappropriate contacts with receptors for other members of this family. These observations also have important caveats for chimera-based protein design because an unexpected gain of function may limit the therapeutic usefulness of some chimeras.
"between FSH (and thyroid-stimulating hormone, TSH) on the one hand, and LH (and chorionic gonadotropin, CG) on the other hand were thought to have separated FSHR-/TSHR-from LHR-activating properties (Campbell et al., 1997; Han et al., 1996). Further separation of specific FSH, TSH and LH activities has been shown by additional sequence divergence in the carboxy-terminal seatbelt segment as well as outside the seatbelt region (Grossmann et al., 1997). "
[Show abstract][Hide abstract] ABSTRACT: Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common alpha subunit non-covalently associated with a hormone-specific beta subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders. Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper. The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review. In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone-receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.
General and Comparative Endocrinology 09/2009; 165(3):412-37. DOI:10.1016/j.ygcen.2009.07.019 · 2.47 Impact Factor
"Like other chimeric proteins, Wld S has a biological activity that requires both of its parts. In other cases, this can be the result of a conformational change that confers a new property such as affi nity for a different receptor in the case of a ligand ( Campbell et al., 1997 ). The combination of two different proteins to form a chimera often arises from chromosome translocations or gene duplications and has evolutionary relevance. "
[Show abstract][Hide abstract] ABSTRACT: The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.
The Journal of Cell Biology 03/2009; 184(4):491-500. DOI:10.1083/jcb.200807175 · 9.83 Impact Factor
"These studies revealed that the region between the 10th and 12th conserved cysteine residues (Cys 10–12 ; i.e. the seat-belt region) of each glycoprotein hormone ␤-subunit is critically involved in determining specificity for its respective receptor. In particular, the net charge differences in the determinant loop (i.e. the region between Cys 10 and Cys 11 ) of the seat-belt region between mammalian LH/CG ␤-subunits on the one hand, and FSH/TSH ␤-subunits on the other hand are thought to have partially separated LH-R-from FSH-R/TSH-R-activating properties (Han et al., 1996; Campbell et al., 1997). It is thought that additional sequence divergence in the carboxy-terminal seat-belt segment (i.e. between Cys 11 and Cys 12 ) as well as outside the seat-belt region has further separated specific lutropic, follitropic and thyrotropic activities of these hormones to their respective natural receptors (Grossmann et al., 1997). "
[Show abstract][Hide abstract] ABSTRACT: Mammalian gonadotropins are highly selective. Charge differences between the Cys(10-11) sequence of FSHbeta and LHbeta/CGbeta seat-belt loops determine the ability of these hormones to interact with the LH-R. Selective FSH-R binding is mainly dependent on the presence of an FSHbeta-specific sequence between Cys(11-12) of the seat-belt loop. Intriguingly, African catfish LHbeta (cfLHbeta) lacks a positively charged Cys(10-11) region and stimulates both catfish LH-R and FSH-R with comparable potencies. Our studies on the promiscuous behaviour of cfLH using chimeric gonadotropins revealed that the Cys(10-11) region of cfLHbeta contains cfLH-R-selective determinants, whereas the Cys(11-12) region of cfLHbeta confers FSH-R-stimulating activity to cfLH. Hence, the location of receptor-selective determinants appeared to be fairly well conserved throughout evolution, despite the low sequence identity between mammalian and catfish seat-belt loops. Moreover, various structure-function differences between gonadotropins are discussed in the context of the different (female) reproductive strategies between mammalian and non-mammalian species that required the divergence to a more specific LH-R-stimulating activity of one of the gonadotropins in mammals.
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