Effects of Binding and Signaling on a  Mutated form of the G-protein Coupled Receptor (GPCR) Thyrotropin Releasing Hormone Receptor

Verietta Williams
Advisor:  Claude Brathwaite, PhD
Avon Foundation Research Program
Cornell University
July 31, 1998
 

Introduction

    G-protein coupled receptors (GPCRs) are membrane bound receptors that carry out some of the signal transduction processes of the cell (2).  They make up 1-2% of the mammalian genome.  They bind a variety of ligands such as ions, lipids, and peptides (3).  GPCRs act as signal transducers by initially interacting with an external ligand.  This signal is then transmitted to the interior of the cell where coupling to the G-protein occurs.  Once the coupling has occurred, other interior effector molecules are activated, such as adenylyl cyclase, ion channels, or phospholipase C.  These final components trigger other second messenger production; this entire process is one of many that regulates the cell (2).
    GPCRs have common structural features.  There is an extracellular domain that has an amino terminus with three loops, a transmembrane portion with seven helices, and an intracellular domain with three loops and a carboxyl terminus (2).  One structural feature of the GPCR that has received attention is the third intracellular loop (I3).  It has been noted that deletions of the third intracellular loop in the GPCR beta-2 adrenergic receptor causes uncoupling of the receptor and the Gs (stimulatory G protein).  The I3 loop is thought to be important for maintenanace of the dormant state of the receptor (6).  Also, it has been observed that a point mutation in the carboxyl terminal in the third intracellular loop of rat thyroptropin stimulating hormone receptor, another G-protein coupled receptor, results in a loss of phosphatidyl-inositol production but there is constitutive signaling activity of the receptor to Gs  (5).
    Constitutive signaling is characterized by a receptorability to signal without the stimulation of an external ligand.  Constitutive signaling of receptors are implicated with cancerous growth.  This phenomenon has been correlated with human disease such as benign toxic thyroid adenomas, thyroid cancer, multinodular goiters, McCune Albright Syndrome, and retinis pigmentosa (2).
 The specific GPCR that was examined this summer was the thyrotropin releasing hormone receptor (TRHR).  Previous work showed the importance of the I3 loop to TRHR coupling and the presence of basal signaling by the receptor.  Present studies looked at the effect of a double mutation R261Q/K262Q in the I3 loop on binding, signaling and basal activity.
 

Materials

Methods

Receptor binding assay

    Binding experiments were performed with intact COS-1 cells in monolayer at 37oC for 1 hour.  TRHR expression (or bound TRH) was measured using varying amounts of unlabeled ligand and [3H] [methyl-His]TRH as radioligand as described (7).  Curves were fit to nonlinear regression analysis and the EC 50 values were quantitated using the PRISM program (GraphPad Inc.)

Inositol phosphate formation

Stimulation of inositol phophate (IP) production was measured in COS-1 cells labeled with myo [3H] inositol which were tested at different concentrations of unlabeled TRH as described (7).  Evaluations of the results were done according to analysis of IP formation that was counted by an automatic counting machine.  These counts were entered into the Prism Graphing Program and examined qualitatively.
 

Luciferase Assay

    Cells were cotransfected with increasing doses of plasmid (10, 40, 80, and 200 ng/ml) and 2 microliters/milliter of pAP1-fos-Luc, which contains the firefly luciferase gene under the control of a protein kinase C-responsive promoter/enhancer (4).  Cells were washed with PBS and lysed with 1X lysis buffer.  Twenty five microliters of each sample was placed in their respective wells in a 96 well plate. Luminometer tubes were placed in conicals with 1X reaction buffer and 1X luciferin.  To each sample was automatically added 125 microliters of reaction buffer and 25 microliters of luciferin and luminescence was measured for 10 seconds.  A direct linear response between transfection plasmid dosage and amount of luminscence was observed in order to get an analysis of wild type plasmid versus mutant plasmid.
 

Results

    Binding--In Figure 1, the most obvious difference between WT and R261Q/K262Q is the TRHR expression level.  Wild type expression is 2.5 times that of R261Q/K262Q.  Our initial results indicate that the KD of WT and R261Q/K262Q seem to be very similar, because, as shown in figure 2, the KD is calculated to be about 15 nM which is close to the wild type KD of 10 nM.
Signaling--In the two experiments done, the signaling ability of R261Q/K262Q was considerably different than wild type.
Basal Activity--A linear increase in luciferase activity is seen with increased transfected plasmid DNA for both WT and R261Q/K262Q (until about 80 ng/ml). Furthermore, there was no binding done to confirm positive TRHR expression along with basal activity.
 

Discussion

    In this study binding, signaling, and basal activity were examined in WT TRHR and R261Q/K262Q TRHR.  The TRHR expression in WT is 5000 dpm whereas the expression in R261Q/K262Q is 2000 dpm.  This is a significant difference.  Calculations reveal that the KD of mutant TRH is about the same in comparison to wild type.  According to luciferase assay results, there seems to be an indication of basal activity in the mutant receptor.  There is a linear increase in luminescence as the transfection plasmid dose increases for wild type.  In R261Q/K262Q this seems to also be the pattern until about 80 ng/ml.  Unfortunately, there needs to be a corresponding binding experiment in order to truly reveal a definite linear increase in TRHR expression along with luminescence.  These results reveal the possibility that the mutant receptor has similar capability for basal activity as the wild type receptor yet it does its work with less receptor expression on the cell membrane.  In conclusion, the amino acid residues 261 and 262 in the I3 loop of TRHR are very important in proper receptor binding, signaling, and basal activity.
 

 References

  1. Arvanitakis, Leandros, Elizabeth Geras-Raaka, and Marvin C. Gershengorn.   Constitutive Signaling G-Protein Coupled Recetpros and Human Disease.  (1998)  Trends in Endocrinology and Metabolism, 9, 27-30.
  2. Berthold, Malin, and Tamas Bartfai.  Modes of Peptide Binding in G Protein Coupled- Receptors.  (1997) Neurochemical Research, 22, 1023-1031.
  3. Jinsi-Parimoo, A. And Marvin C. Gershengorn.  (1997)  Endocrinology 138, 1471- 1475.
  4. Lefkowitz, R.J., Susanna Cotecchia, Philippe Samama, and Tommaso Costa. Constitutive activity of receptors coupled to guanine nucleotide regulatory  proteins. (1993) Trends in Pharmacological Science.  14, 303-307.
  5. Strader, C.D., et al.  G-Protein Coupled Receptors.  (1994)  Annual Review of  Biochemistry.  63, 101-132.
  6. Straub, R.E., Rech, G. C., Joho, R.H., and Gershengorn, M.C.  (1990) Proceedings in  the National Academy of Science U.S.A.  87, 9514-9518.