Statement of research interests:

My primary research focus is continuing my previous work on the human follicle stimulating hormone receptor to understand how the signaling pathway is regulated.  However, I have many other research topics. More information about them can be found here.

G protein-coupled receptor signaling and desensitization

G protein-coupled receptors (GPCR) are transmembrane receptors that are involved in varied physiological processes ranging from reproduction to vascular constriction (1).  Some estimates suggest that GPCR and their signaling pathways are the targets of as much as 50% of all pharmaceuticals (2).  Therefore, understanding how these receptors function and how they are regulated are important questions in a vast range of disciplines.

Human follicle
Text Box: Figure 1. Schematic of human follicle stimulating hormone receptor. A) Extracellular ligand binding domain. B)Transmembrane domain. (2).

Background

During my postdoctoral work, I investigated the role of accessory proteins in human follicle stimulating hormone receptor (hFSHR) regulation and the role of ubiquitination in receptor internalization (3-6).  hFSHR is involved in spermatogenesis in males and oocyte development in females and represents an important therapeutic target for treating infertility (3).  A member of the glycoprotein hormone receptor family, hFSHR has unique features which make it a good model to study receptor desensitization.  In particular the receptor has a large extracellular domain where ligand binding takes place (figure 1).  I propose to remove the extracellular domain of the receptor and employ a wide assortment of molecular and biochemical techniques to investigate signaling and desensitization of the truncated receptor.  Creating a mutant of the receptor which cannot bind ligand will allow us to observe if ligand-independent receptor desensitization takes place.

Rationale

The idea of ligand-independent desensitization has its roots in the “spare receptor” hypothesis (7, 8).  Since receptor signaling reaches a maximum before binding of ligand reaches its maximum, it was hypothesized that extra receptors on the cell surface were actively desensitized coordinately with the bound receptors as a means of preventing additional signaling.  This would require phosphorylation of the receptor without a signal having been sent.  This is inconsistent with standard models of hFSHR activation and signal termination but precedent for this model can be found in the example of the histamine H1 receptor (9).  The challenge is to distinguish between receptors that were desensitized because they were occupied and receptors which were naïve and had never seen ligand on the same cell.

Previous Work

We have constructed a mutant hFSHR which is incapable of binding ligand because the entire ligand binding domain has been removed.  This truncated receptor has been modified to include an N-terminal signal sequence to allow for secretion to the cell surface and a “FLAG” peptide tag for detection with antibodies.

Research Plan

Mutant and wild type receptors will be expressed in HEK-293 cells, a model mammalian cell line which has been used previously to investigate mutant hFSHR expression (4).  Transfection of expression plasmids into cells will be followed by validation of protein expression by immunofluorescene microscopy and western blotting.  The goal is to co-express the truncated receptor with the wild type form and determine if the truncated receptor is phosphorylated.  This can be detected by immunoprecipitating the truncated receptor and detecting phosphorylation using phosphoserine and phosphothreonine specific antibodies.  Students will have the opportunity to develop and apply skills in microscopy, cell culture and protein expression, as well as protein gels and western blots.

What We Will Learn

Detection of phosphorylation will allow us to ask additional questions about the kinases responsible for phosphorylating the hFSHR, the role of G protein coupling in preventing premature desensitization, and the role of receptor dimerization and aggregation in lipid rafts in ligand-independent phosphorylation.   However, in the absence of ligand independent phosphorylation, other opportunites can be developed.  Why does signaling maximize before receptor occupancy?  Is effector limiting or is sequestering of receptor responsible for termination of signaling?  Does ligand-independent internalization take place as receptor gets sequestered into clathrin coated pits?  These investigations into basic GPCR physiology can be asked in tandem with questions about ligand-independent internalization.

References

  1. Benovic JL, Onorato JJ, Caron MG, Lefkowitz RJ 1990 Regulation of G protein-coupled receptors by agonist-dependent phosphorylation. Soc Gen Physiol Ser 45:87-103
  2. Ja WW, Roberts RW 2005 G-protein-directed ligand discovery with peptide combinatorial libraries. Trends Biochem Sci 30:318-324
  3. Dias JA, Cohen BD, Lindau-Shepard B, Nechamen CA, Peterson AJ, Schmidt A 2002 Molecular, structural, and cellular biology of follitropin and follitropin receptor. Vitam Horm 64:249-322
  4. Cohen BD, Bariteau JT, Magenis LM, Dias JA 2003 Regulation of follitropin receptor cell surface residency by the ubiquitin-proteasome pathway. Endocrinology 144:4393-4402
  5. Cohen BD, Nechamen CA, Dias JA 2004 Human follitropin receptor (FSHR) interacts with the adapter protein 14-3-3tau. Mol Cell Endocrinol 220:1-7
  6. Nechamen CA, Thomas RM, Cohen BD, Acevedo G, Poulikakos PI, Testa JR, Dias JA 2004 Human follicle-stimulating hormone (FSH) receptor interacts with the adaptor protein APPL1 in HEK 293 cells: potential involvement of the PI3K pathway in FSH signaling. Biol Reprod 71:629-636
  7. Marunaka Y, Niisato N, Miyazaki H 2005 New concept of spare receptors and effectors. J Membr Biol 203:31-39
  8. Homer LD, Nielsen TB 1987 Spare receptors, partial agonists, and ternary complex model of drug action. Am J Physiol 253:E114-21
  9. Iwata K, Luo J, Penn RB, Benovic JL 2005 Bimodal regulation of the human H1 histamine receptor by G protein-coupled receptor kinase 2. J Biol Chem 280:2197-2204