[Frontiers in Bioscience 11, 1522-1539, May 1, 2006]

Regulation of Protein 4.1R Interactions with Membrane Proteins by Ca2+ and Calmodulin

Wataru Nunomura and Yuichi Takakuwa

Department of Biochemistry, Tokyo Women's Medical University, 8-1 Kawada, Shinjuku, Tokyo 162-8666, Japan

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Techniques Used for Measuring Binding Affinity
4. Organization of the Erythrocyte Membrane Skeleton
4.1. Maintenance of Erythrocyte Shape
4.2. Maintenance of Erythrocyte Membrane Mechanical Properties
5. 4.1R as a Modulator in a Complex Membrane System
5.1. Facilitation of p55 Binding to GPC
5.2. Modulation of Ankyrin Binding to Transmembrane Proteins
5.3. Facilitation of Spectrin/Actin Interaction
6. Ca2+/CaM-Dependent Regulation of 4.1R Interactions
6.1. Variations in Intracellular Ca2+ Level
6.2. 4.1R, a CaM-Binding Protein with Unique Features
6.3. Ca2+/CaM-Dependent Regulation of Membrane Protein Interactions
7. Functional Structure of 4.1R
7.1. Overview of 4.1R 7.2. 30kDa (FERM) Domain
7.2.1. Crystal Structure of 4.1R 30kDa (FERM) domain
7.3. 10kDa (SAB) Domain
7.4. 22/24kDa (CTD) Domain
8. At least Two Alternative Splicing Initiation Sites in One Single Gene
9. 4.1R homologues
10. Conclusion
11. Appendix
12. Acknowledgements
13. References

1. ABSTRACT

Red blood cell protein 4.1 (4.1R) is essential for maintaining erythrocyte shape and controlling membrane mechanical properties, such as deformability and stability. The importance of 4.1R has been demonstrated by the dramatic erythrocyte alterations observed in patients lacking this protein. Indeed, 4.1R null red blood cells adopt an elliptical shape and are characterized by unstable membranes. The key role of 4.1R likely results from multiple protein-protein interactions: lateral interactions with the spectrin/actin network and vertical interactions with the cytoplasmic domain of transmembrane proteins glycophorin C (GPC), Band 3 (anion exchanger 1, AE1), and CD44. 4.1R promotes the formation of a ternary complex with GPC and p55 through its 30kDa membrane-binding domain. Based on the primary structure of the prototypical 80kDa isoform of 4.1R, functional domains and sites for binding partners have been identified. The others and we have been focusing on the structure and function of the 30kDa NH2-terminal domain of 4.1R, which is responsible for 4.1R interaction with the transmembrane proteins described above. A major finding is that Ca2+, in association with calmodulin (CaM), plays a critical role in regulation of the interaction of the 30kDa domain with its various binding partners. This review is a detailed report of our current knowledge regarding 4.1R, and more specifically, 4.1R 30kDa domain: its primary structure, functions and modulation by Ca2+ and CaM. Emphasis is given on the relationships between structure and function that we have been able to establish through X-ray crystal structure analysis of the 30kDa membrane-binding domain in 4.1R. Finally, we give insights into the potential roles of 4.1R in the dynamic organization of the membrane skeleton viewed as a complex system.