Amino Acid Residues Implicated in the Interaction of Melanocortin Ligands and their Receptors: A Study of MC2R Selectivity

Author: Kristopher D. Veo Title: Amino acid residues implicated in the interaction of Melanocortin ligands and their receptors: A study of MC2R selectivity Advisor: Dr. Robert M. Dores Degree Date: August 2009 ABSTRACT Melanocortin receptor ligand selectivity has been a question not easily answered. The inability to functionally express melanocortin 2 receptor (MC2R) has inhibited the study of why MC2R is only stimulated by ACTH, a melanocortin hormone. With the recent discovery of the MC2R accessory protein (MRAP), creating a heterologous system is now feasible. Using a general cell line like CHO-K1 cells, which do not express endogenous MCRs, we were able to create a heterologous expression system and test the selectivity of MC2R using analog variants of ACTH(1-24). Our results indicate an amino acid requirement in the C-terminal portion of ACTH(1-24) for activation, which supports the 2-step method of activation hypothesized for MC2R. This site, the tetra basic cleavage site, when altered does not stimulate cAMP production and does not compete with ACTH(1-24) for binding. We also demonstrate the potential for a non-mammalian MC2R system in cloning full length Silurana tropicalis MC2R and completed localization studies with this system with MRAP using CHO-K1 cells.

Evolution of Melanocortin-2 Receptor Activations: Studies on a Mammal and a Fish

The structure and function relationship between melanocortin-2 receptor (MC2R) and ACTH are the most complicated in melanocortin receptor gene family. A comparative study on the activation of human and rainbow trout MC2R will provide a useful model system for understanding how ACTH emerged as the sole ligand for the MC2R of bony vertebrates. This dissertation will discuss how studies utilizing analogs of hACTH(1-24) have revealed two critical amino acid motifs in this ligand (HFRW and KKRRP) which are required for the activation of MC2R. In addition, the KKRRP motif functioned as the unique binding site for MC2R that directly contributes to the ligand selectivity feature, as revealed from studies on an ACTH antagonist which exclusively targets MC2R. Finally, based on our model for the interaction of ACTH and MC2R, the amino acid residues within TM4, EC2, and TM5 domains responsible for ACTH ligand selectivity will be evaluated by site-directed mutagenesis studies.

Parent-Child Interaction and Children's Self-Concept

This study is designed to investigate the relationships between marital communication, the quality of parents' ability to assist their children in joint problem-solving, and children's independent mastery attempts and perceived competence at problem-solving, and behavioral indicators of self-esteem. Couples' skill at regulating their own and their children's negative affect within the marital and parent-child family subsystems is hypothesized to predict the quality of their assistance, or scaffolding behavior, to their children during joint problem-solving. Further, the quality of parental scaffolding behavior is expected to predict children's independent mastery attempts, levels of perceived competence at problemsolving, and behavioral indicators of self-esteem. Families for the study will be those with children between 3 1/2 to six years of age recruited from subjects participating in a longitudinal study of communication in marriage being conducted at the Denver Center for Marital and Family Studies. Families will participate in three interaction tasks designed to tap parental scaffolding behavior during problemsolving with their children. Children will be administered self-report measures to tap their perceived competence at such problem-solving as those in the interaction tasks and parents will complete a questionnaire tapping the behavioral indicators of their child's self-esteem. Family interaction data will be coded with the use of a microanalytic coding system developed by this study, the Parent-Child Interaction Coding System. Marital communication data at three time points, premaritally, during the transition to parenthood , and concurrently, will be obtained from couples' interactions from the longitudinal study. The clinical significance of this study includes implications for training couples how to effectively regulate negative affect and offer their children sensitive assistance during joint problem-solving.