Subunits of luteinizing hormone-human chorionic gonodotropin receptor from bovine corpora lutea

Abstract

A batch of 24 mg of luteinizing hormone-human chorionic gonadotropin (LH-hCG) receptor was isolated from bovine corpora lutea. The LH-hCG receptor showed specific binding with hCG. The receptor-hCG complex activated the regulatory Ns protein isolated from rabbit liver, which in turn stimulated adenylate cyclase to convert ATP into cAMP in vitro, attesting to the biological activity of the purified LH-hCG receptor. The LH-hCG receptor was treated with 2% sodium dodecyl sulfate (SDS) to prepare the molecular weight (Mr) 280K dimer and with 50 mM dithiothreitol (DTT) to prepare the Mr 120K monomer and subunits of Mr 85K and 38K. Oligomers of various molecular weights were recovered from gel filtration columns due to the reassociation of disulfide bonds between monomers and subunits. Hence, the receptor monomer was also dissociated into subunits of Mr 85K and 38K by reduction of -S-S-bonds with 50 mM DTT in 2% SDS and alkylation of sulfhydryl groups in the presence of 100 mM N-ethyl-maleimide. The subunits were separated by gel filtration through columns of Ultrogel AcA-44 and Sephadex G-75. The yields of the purified alkylated subunits of Mr 85K and 38K were 1.8 and 1.5 mg, respectively. Each subunit migrated as a single entity in SDS-polyacrylamide gel electrophoresis. The monomer of the receptor of Mr 120K showed specific binding with 125I-hCG, suggesting it to be the minimum molecular weight functional unit of the receptor. The Mr 85K and 38K subunits bound 125I-hCG, which could not be displaced with unlabeled hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

Corpus Luteum Function Following Spontaneous or Prostaglandin-Induced Estrus in Brahman Cows and Heifers

“…Even though luteal cell populations were altered by PGF and age of animal, existence of spare (unoccupied) receptors may have masked any influence of PGF or age on total LH receptors. Unoccupied receptor concentration ranged from .75 to 2.0 fmol/mg wet weight, which was similar to results (bovine) reported by Rutter et al (1985) and Spicer et al (1981), but was lower than results (bovine) reported by Saxena et al (1984). The lower receptor concentration observed in this study could be due to the animal model utilized: Bos indicus cattle have smaller CL that are less steroidogenically functional than Bos taurus cattle (Irvin et al, 1978;Segerson et al, 1984).…”

Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary.

“…For example, whereas Kusuda and Dufau (10) identified the purified LH/hCG receptor as a single protein of Mr 73,000, Bruch et al (11) reported that the purified receptor consists of four nonidentical subunits of Mr 79,300, 64,400, 55,300, and 46,700, although in a subsequent study it was concluded that the receptor may exist as a single polypeptide (14). Saxena et al (12) reported that a Mr 120,000 protein represents the functional unit of the hCG receptor and that this Mr 120,000 protein is composed of Mr 85,000 and 38,000 proteins linked covalently through disulfide linkages. More recently, Rebois et al (8), on the basis of hydrodynamic properties, reported that the receptor is composed of two dissimilar subunits held together noncovalently: a hormonebinding subunit of Mr 76,000 and a Mr 50,000 component.…”

“…Reports have appeared regarding the structure of the ovarian LH/CG receptor as determined by chemical crosslinking (2-4), by photoaffinity labeling (5-7), and by flotation in sucrose density gradients combined with gel filtration chromatography (8). Recently, more information about the ovarian LH/CG receptor has resulted from attempts by several laboratories to purify the receptor by affinity chromatography (9)(10)(11)(12)(13)(14)(15). The conclusions of these studies suggested that the receptor could be either a heteropolymer or a single polypeptide.…”

“…The conclusions of these studies suggested that the receptor could be either a heteropolymer or a single polypeptide. However, there appears to be a consensus that the molecular weight of one of the binding domains of the receptor is in the range of70,000-95,000 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). In the present studies we have examined the possible subunit structure of the affinity-purified receptor by a combination of SDS/ PAGE, direct ligand blotting, and electroelution.…”

Factors affecting reproductive efficiency of cattle

“…Human chorionic gonadotropin, derived from the chorion, is LH-like and preferentially binds to the α subunit of the LH receptor (Hong et al, 1999). When the receptor complex, located on the small luteal cells (Mamluk et al, 1998), is bound by either hCG or LH, it acts through the adenylate cyclase pathway, catalyzing the conversion of ATP to cAMP (Saxena et al, 1986). Some actions of hCG include a luteotropic effect similar to LH (Donaldson and Hansel, 1965) and stimulation of synthesis of progesterone by the CL in vitro (Veenhuizen et al, 1972) and in vivo (Breuel et al, 1989).…”