Research

The Glycoprotein hormones are the key hormones regulating reproduction and overall physiology of the organism. These hormones and their receptors are also dream molecules for an adventurous protein chemist. The laboratory has been investigating these hormones for their physiological roles, molecular mechanism of action, structures and biotechnological applications.

The Glycoprotein hormones, Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) produced by the pituitary and human Chorionic Gonadotropin (hCG) produced by the placenta are heterodimeric proteins with an identical a subunit associated noncovalently with the hormone specific b subunit. Both subunits are glycosylated and glycosylation during biosynthesis is absolutely essential for right folding. of each subunit.

Both subunits have several disulfide bridges and the crystal structures of hCG and human FSH reveal that three of the disulfide bridges in both subunits form cystine knots. The receptors for these hormones belong to the family of G-protein coupled receptor family with a large extracellular domain (ECD) believed to be involved in specific hormone binding and a characteristic seven transmembrane domain (TMD) involved in signal transduction.

The ultimate goal of the laboratory is to understand the mechanism of action of these hormones at the molecular level and our specific aims are understand how these hormones bind to their specific receptor and decipher the mechanism of signal transduction. For this, we have employed variety of strategies and generated different tools. The laboratory has hyperexpressed, purified and characterized all the four human Glycoprotein hormones, some superagonists and antagonists by recombinant DNA technology. Availability of purified recombinant hormones and their analogs has encouraged us to explore the possibility of using these molecules in clinical situations such as treatment of infertility or reproductive abnormalities, thus leading to interactions with biotechnology companies. At the same time, attempts are now being made to crystallize the analogs of the hormones and determine their structures. We are also labeling the recombinant proteins with 15N which will enable us to undertake a rather ambitious programme of determining the solution structures of the glycoprotein hormones and their receptors by NMR spectroscopy.

Antibodies have proved to be excellent tools for identification of the contact points between hormones and their receptors. Our studies have demonstrated that the initial interaction between the two entities is through the hormone specific b subunit and the extracelluar domains of the receptors which results in bringing the common a subunit in contact with the transmembrane domain leading to signal transduction.

The ECDs of the Glycoprotein hormone receptors are divided into two distinct domains, the N terminal domain with several Leucine Rich Repeats (LRR) and studies including determination of crystal.structures of ECD-hormone complexes have shown that the hormones actually bind to this part of the receptors, but this alone is not enough for signal transduction. Our studies with antibodies to different LRRs, as well as, to the distinct epitopes of the hormones are in complete agreement with the crystal structure data.

 

 

The second part of the receptor called the ‘Hinge region’, which connects the LRR to the TMD, has proved to be very exciting as it is now believed that this region is probably involved in the signal transduction process. Our recent data show that an antagonist of the hormone interacts with this portion of the receptor and inhibits hormone action. Using highly characterized polyclonal, monoclonal and recombinant antibodies to different stretches of the receptor we are now trying to identify the changes in the conformation of the receptors once hormones bind to their receptors

We have also investigated FSHb gene expression. Our studies demonstrated the structure of mRNA regulates FSHb gene expression. We identified elements within the coding portion of FSHb mRNA (Samaddar et al, Gene 1999) and in 3’ Untranslated region of the mRNA (Manjithaya and Dighe; Biology of Reproduction, 2004) that are responsible for the down regulation of FSHb expression.

The research pursued in the laboratory has several applications. The wide ranging expertise available in the laboratory is being used to develop novel strategies for diagnosis and treatment of infertility, reproductive failures, thyroid abnormalities and other endocrine disorders. A new initiative that is being launched is to characterize Indian infertile patients and study the genetics of human infertility by investigating mutations in the hormone and their receptors. It is expected that the analogs of the hormones and antibodies will have direct applications in treating infertility and abnormal reproductive processes such as precocious puberty.

In addition, attempts are already underway to hyperexpress, purify and characterize other human hormones such as human Growth hormone and insulin. Research in fermentation technology has already allowed us to scale production of the recombinant hormones which can be transferred to biotech industries for production of clinical grade hormones. Simpler and quicker methods are being developed for purification of the recombinant hormones. These studies are also expected to bring down the cost of hormone treatment in India which at the moment is prohibitively expensive. The laboratory interacts extensively with biotech industries for developing new recombinant proteins, diagnostic tools and assay procedures.

 

Research Themes for potential students:

 

Structures of Glycoprotein hormones and their analogs with potential clinical applications and their interactions with receptors

 

Glycoprotein hormones receptors and the mechanism of receptor activation with a possible link to infertility, other reproductive disorders and diseases such as cancer. 

 

Hormonal regulation and Molecular biology of spermatogenesis.

The antibodies against Gonadotropins were also employed to conclusively resolve a hotly debated topic in the field of male reproduction of whether FSH has a role in regulation of the adult spermatogenesis (Shetty et al, Endocrinology, 1996; Marathe et al, Endocrine, 1995). Our data demonstrated that specific blocking of either LH or FSH action results in apoptotic cell death of testicular germ cells, thus providing a clue to the biochemical actions of these hormones in the regulation of spermatogenesis (Joshi and Dighe, J. Steroid Biochemistry and Molecular Biology,2006; Pareek et al, Apoptosis, 2007). Our recent efforts have been directed towards understanding the molecular biology of human spermatogenesis and gaining insights into the transcriptomes of human tetraploid and haploid cells and understanding the transcriptional regulation of the genes expressed differentially during spermatogenesis. We have shown that SRY, a gene encoded by the Y chromosome and known to be the critical factor for development of male reproductive system, is one of the key regulators of gene expression in haploid cells (Sanyal et al, manuscript in preparation).

E12 is one of the Mab that can uniquely recognize hetero dimeric hCG  with no cross reactivity to LH

The schema of immunomapping , mapping  of protein-protein interaction using antibodies or ScFvs. Shown here is the Immune mapping of hCG and its receptor has helped predict hormone receptor interaction dynamics.

Simulation of hormone receptor interaction can be carried out using antibodies. RF2 antibody gives critical insights on the signal transduction mechanism of GpH receptors and GPCRs in general.

Treatment with GnRH antagonist or administration of neutralizing antibodies against LH and FSH bring about apoptosis of the germ cells. Thus, the Gonadotropins play important roles in the survival of the testicular germ cells .

Interestingly, the recent data from the laboratory have also shown that some of the antibodies specific for the hinge region simulate hormone action further confirming the key role played by this region is signal transduction. This very exciting observation is now being explored further with monoclonal and recombinant antibodies. The experimental strategies we are using are

· Expression and purification of functional ECDs of human LH, FSH and TSH receptors

· Immunological mapping of ECDs using polyclonal, monoclonal and recombinant antibodies

· Biochemical analysis using cell lines expressing glycoprotein hormone receptors

· Mutational analysis of receptors

· Structural biology approach of investigating the structures of hormone receptors either by crystallography or NMR spectroscopy, this being part of international collaborative efforts

Prof. Rajan R. Dighe’s

Laboratory

Structure function analysis lab

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