Cloning, sequencing and in vitro functional expression of recombinant donkey follicle-stimulating hormone receptor: a new insight into the binding specificity of gonadotrophin receptors.

The study involves the research about a specific receptor (FSH-R) found in donkeys and its binding characteristics with certain substances. Researchers cloned the receptor from donkey testis mRNA and studied its ability to bind to horse LH/CG and FSH as well as generate cAMP when FSH is present. The structure of the donkey receptor was found to be closely similar to the horse receptor, differing only in four amino acids. These differences may play a part in preventing LH/CG from binding to FSH-R.

Cloning and Sequencing of Donkey FSH Receptor

• The research starts with the cloning of the donkey Follicle Stimulating Hormone Receptor (FSH-R), a type of protein found on the surfaces of cells that responds to the presence of FSH, a hormone involved in the reproductive processes of mammals.
• To get a template for the cloning, the researchers extracted messenger RNA, which carry genetic information, from donkey testis and reverse transcribed it into complementary DNA (cDNA).
• The cloned cDNA was then sequenced to determine the specific order of nucleotides, which define the properties and functions of the resulting protein.

In vitro Functional Expression of Donkey FSH Receptor

• Once the donkey FSH-R was cloned and sequenced, the researchers moved on to study its functions in vitro, meaning within a controlled laboratory setting.
• Part of this functional expression study involved examining what hormones or substances the donkey FSH-R can bind to.
• The cloned donkey FSH-R when introduced to COS-7 cells revealed both horse LH/CG and FSH binding activity, and was even capable of stimulating cAMP production when FSH was present.

Amino Acid Differences and Binding Specificity

• The researchers noted that the sequence of the cloned donkey FSH-R showed 96% similarity with horse FSH-R, meaning that they are largely identical and likely have similar functions.
• The remaining 4% difference in the sequences is due to the presence of four divergent amino acids, namely: Thr 173, Asp 202, Asn 268 and Pro 322.
• These amino acid discrepancies could impact the binding specificity of the receptor, potentially inhibiting LH/CG from binding to the FSH-R.
• Specifically, Asn 268 in horse FSH-R carries an additional N-glycosylation, a process where a carbohydrate is added to a protein, which could further contribute to the binding specificity.